Allosteric modifiers of hemoglobin useful for decreasing oxygen affinity and preserving oxygen carrying capability of stored blood

ABSTRACT

A family of compounds has been found to be useful for right-shifting hemoglobin towards a low oxygen affinity state. The compounds are capable of acting on hemoglobin in whole blood. In addition, the compounds can maintain the oxygen affinity in blood during storage and can restore the oxygen affinity of outdated blood.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application is a continuation-in-part (CIP) application ofSer. No. 08/101,501, dated Jul. 30, 1993 and which issued as U.S. Pat.No. 5,432,191, which in turn is a C-I-P of Ser. No. 08/006,246 filedJan. 19, 1993, now U.S. Pat. No. 5,290,803, which in-turn is a C-I-P ofSer. No. 07/722,382, filed Jun. 26, 1991, now U.S. Pat. No. 5,382,680,which in-turn is a C-I-P of Ser. No. 07/702,947 filed May 20, 1991,which in-turn is a C-I-P of Ser. No. 07/478,848 filed Dec. 12, 1990,U.S. Pat. No. 5,049,695, which in-turn is a continuation of Ser. No.07/623,346 filed Dec. 7, 1990, now abandoned.

DESCRIPTION BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention generally relates to using a family of compoundsto adjust the allosteric equilibrium of hemoglobin toward a low oxygenaffinity state. Moreover, the invention includes several new compoundsand contemplates using the family of compounds for use in treatingdiseases involving oxygen deficiency, in wound healing, and in restoringoxygen affinity of stored blood.

2. Description of the Prior Art

Hemoglobin is a tetrameric protein which delivers oxygen via anallosteric mechanism. Oxygen binds to the four hemes of the hemoglobinmolecule. Each heme contains porphyrin and iron in the ferrous state.The ferrous iron-oxygen bond is readily reversible. Binding of the firstoxygen to a heme requires much greater energy than the second oxygenmolecule, binding the third oxygen requires even less energy, and thefourth oxygen requires the lowest energy for binding. Hemoglobin has twoα and two β subunits arranged with a two fold symmetry. The α and βdimers rotate during oxygen release to open a large central watercavity. The allosteric transition that involves the movement of thealpha-beta dimer takes place between the binding of the third and fourthoxygen. The α₁ β₁ interface binding is tighter than the α₁ α₂ or α₁ β₂interfaces.

In blood, hemoglobin is in equilibrium between two allostericstructures. In the "T" (for tense) state, hemoglobin is deoxygenated. Inthe "R" (for relaxed) state, hemoglobin is oxygenated. An oxygenequilibrium curve can be scanned, using well known equipment such as theAMINCO™ HEM--O--SCAN, to observe the affinity and degree ofcooperativity (allosteric action) of hemoglobin. In the scan, the Y-axisplots the percent of hemoglobin oxygenation and the X-axis plots thepartial pressure of oxygen in millimeters of mercury (mmHg). If ahorizontal line is drawn from the 50% oxygen saturation point to thescanned curve and a vertical line is drawn from the intersection pointof the horizontal line with the curve to the partial pressure X-axis, avalue commonly known as the P₅₀ is determined (i.e., this is thepressure in mmHg when the scanned hemoglobin sample is 50% saturatedwith oxygen). Under physiological conditions (i.e., 37° C., pH=7.4, andpartial carbon dioxide pressure of 40 mm Hg), the P₅₀ value for normaladult hemoglobin (HbA) is around 26.5 mmHg. If a lower than normal P₅₀value is obtained for the hemoglobin under test, the scanned curve isconsidered to be "left-shifted" and the presence of high affinityhemoglobin is indicated. Conversely, if a higher than normal P₅₀ valueis obtained for the hemoglobin under test, the scanned curve isconsidered to be "right-shifted" and the presence of low affinityhemoglobin is indicated.

It has been proposed that influencing the allosteric equilibrium ofhemoglobin is a viable avenue of attack for treating diseases. Theconversion of hemoglobin to a high affinity state is generally regardedto be beneficial in resolving problems with deoxy Hemoglobin-S (sicklecell anemia). The conversion of hemoglobin to a low affinity state isbelieved to have general utility in a variety of disease states wheretissues suffer from low oxygen tension, such as ischemia and radiosensitization of tumors. Several synthetic compounds have beenidentified which have utility in the allosteric regulation of hemoglobinand other proteins. For example, several new compounds and methods fortreating sickle cell anemia which involve the allosteric regulation ofhemoglobin are reported in U.S. Pat. No. 4,699,926 to Abraham et al.,U.S. Pat. No. 4,731,381 to Abraham et al., U.S. Pat. No. 4,731,473 toAbraham et al., U.S. Pat. No. 4,751,244 to Abraham et al., and U.S. Pat.No. 4,887,995 to Abraham et al. Furthermore, in both Perutz, "Mechanismsof Cooperativity and Allosteric Regulation in Proteins", QuarterlyReviews of Biophysics 22, 2 (1989), pp. 163-164, and Lalezari et al.,"LR16, a compound with potent effects on the oxygen affinity ofhemoglobin, on blood cholesterol, and on low density lipoprotein", Proc.Natl. Acad. Sci., USA 85 (1988), pp. 6117-6121, compounds which areeffective allosteric hemoglobin modifiers are discussed. In addition,Perutz et al. has shown that a known antihyperlipoproteinemia drug,bezafibrate, is capable of lowering the affinity of hemoglobin foroxygen (see, "Bezafibrate lowers oxygen affinity of hemoglobin", Lancet1983, 881.

German Patent Applications 2,149,070 and 2,432,560, both to Witte etal., disclose compounds which are structurally similar to some of thecompounds in the family of compounds defined by this invention. However,the Witte et al. patent applications contemplate use of the compoundsfor the reduction of serum lipid levels. The Witte et al. patentapplications do not provide any indication of the potential use of thecompounds for allosteric hemoglobin modification.

SUMMARY OF THE INVENTION

It is therefore an object of this invention to provide a method of usinga family of compounds to allosterically modify hemoglobin such that thehemoglobin is present in blood in a lower oxygen affinity state.

It is another object of this invention to provide a method of prolongingthe storage life of blood by adding compounds within a particular familyof compounds to the blood.

It is another object of this invention to provide new compounds whichare capable of allosterically modifying hemoglobin.

According to the invention, an allosteric hemoglobin modifying family ofcompounds is defined by the formula:

    R.sub.1 --(A)--R.sub.2

where R₁ and R₂ each are a substituted or unsubstituted aromatic orheteroaromatic compound, or substituted or unsubsituted alkyl orheteroalkyl ring compound, or a substituted or unsubstituted phthalimidecompound, and where R₁ and R₂ may be the same or different, where A is achemical bridge which includes two to four chemical moieties bondedtogether between R₁ and R₂, wherein said chemical moieties in A areselected from the group consisting of CO, O, S, SO₂, NH, NR₃ where R₃ isa C₁₋₆ alkyl group, NR₄ where R₄ includes two carbonyls as part of aphthalimide compound formed with R₁ or R₂, CH₂, CH, and C, with thecaveat that, except in the case where A contains two identical CH and Cmoieties positioned adjacent one another to form an alkene or alkyne,the chemical moieties in A are each different from one another, andwherein at least one of R₁ or R₂ is substituted with a compound havingthe chemical formula: ##STR1## where n is zero to five, where R₅ and R₆are selected from the group consisting of hydrogen, halogen, substitutedor unsubstituted C₁₋₁₂ alkyl groups, and substituted or unsubstitutedaromatic or heteroaromatic groups, and these moieties may be the same ordifferent, or alkyl moieties of part of an aliphatic ring connecting R₅and R₆, and where R₇ is a hydrogen, halogen, salt cation (e.g., sodium,potassium, ammonium, etc.), metal, or substituted or unsubstituted C₁₋₆alkyl group.

Many compounds within this family have been synthesized and their effecton the P₅₀ value of hemoglobin has been determined. Each of thecompounds tested was found to increase the P₅₀ value of hemoglobin;hence, the compounds are capable of driving the allosteric equilibriumof hemoglobin towards a condition favoring the low oxygen affinitystate. In addition, the compounds were found to stabilize the degree ofoxygen dissociation of hemoglobin in stored blood over extended periodsof time. Furthermore, the compounds were found to be well tolerated bymice when administered as an intraperitoneal dose. Because the compoundswithin the family defined by this invention are capable of shifting thehemoglobin allosteric equilibrium toward the low affinity "T" state,they have the ability to cause hemoglobin to deliver more oxygen totissues. Thus, the compounds of the invention should be valuable asantiischemic agents, as sensitizers for x-ray irradiation in cancertherapy, as wound healing agents, in treating disorders related to lowoxygen delivery in the brain such as Alzheimer's, depression, andschizophrenia, in preparing blood substitutes, and in blood storage, aswell as other applications.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects, aspects and advantages will be betterunderstood from the following detailed description of a preferredembodiment of the invention with reference to the drawings, in which:

FIG. 1a is a chemical structure defining a particularly preferred groupwithin the family of compounds used in the present invention;

FIGS. 1b and 1c are chemical structures defining two subsets of thefamily defined in FIG. 1a;

FIGS. 2a-b depict chemical structures of precursor compounds arranged inreaction schemes for preparing compounds that are useful asintermediates for synthesizing compounds within a first group of thefamily of compounds;

FIG. 2c depicts chemical structures, including the intermediatesproduced as shown in FIGS. 2a-b, arranged in a reaction scheme toprepare the first group of preferred compounds;

FIG. 3 depicts chemical structures arranged in a reaction scheme toproduce a second group of the family of preferred compounds;

FIG. 4 depicts chemical structures arranged in a reaction scheme toproduce a third group of the family of preferred compounds;

FIG. 5a-b depict chemical structures of precursor compounds arranged inreaction schemes for preparing compounds that are useful asintermediates for synthesizing compounds within a fourth group of thefamily of preferred compounds;

FIG. 5c depicts chemical structures, including the intermediatesproduced in FIGS. 5a-b, arranged in a reaction scheme to produce thefourth group of compounds;

FIG. 6a depicts chemical structures arranged in a reaction scheme, whichis an alternative to that shown in FIG. 4, for producing compoundswithin a third group of the family of preferred compounds;

FIG. 6b depicts chemical structures arranged in a reaction schemesimilar to that shown in FIG. 6a, except that the precursor compoundsutilized are chosen such that the compound produced has ameta-substitution rather than para-substitution on one phenyl ring andso that ethyl rather than methyl groups are present on the substituentmoiety of the meta-substituted phenyl ring;

FIG. 6c depicts chemical structures arranged in a reaction schemewhereby groups larger than ethyl are introduced into the acid moiety ofcompounds of Group III;

FIGS. 7a and 7b depict chemical structures arranged in a reaction schemefor producing compounds within a fifth group of the family of preferredcompounds;

FIG. 8 is a graph illustrating the oxygen dissociation curves producedwhen a 5.4 millimolar solution of normal hemoglobin in the presence andabsence of selected compounds is tested at pH 7.4 using HEPES as thebuffer in a Hem-O-Scan oxygen dissociation analyzer;

FIG. 9 is a graph similar to FIG. 8 which illustrates oxygendissociation curves for whole human blood in the presence and absence ofselected compounds;

FIG. 10 is a graph similar to FIG. 8 where the oxygen dissociationcurves produced are for a 5.4 millimolar solution of normal hemoglobinin the presence and absence of particular compounds, including2,3-diphosphoglycerate which is the natural allosteric hemoglobineffector, are tested at pH 7.4 using HEPES as the buffer in a Hem-O-Scanoxygen dissociation analyzer;

FIG. 11 is a bar graph showing the percentage oxygen delivered by packedcells, fresh, stored and in the presence of 2- 4-((((3,5-dimethylphenyl)amino)carbonyl)methyl)phenoxy!-2-methyl propionic acid (C₂₀ H₂₃ NO₄),respectively;

FIGS. 12a-b depict chemical structures in a reaction scheme used toproduce a phthalimide form of the compounds within the present inventionwhere the compounds were shown by a measured P₅₀ value to allostericallymodify hemoglobin towards the low oxygen affinity state;

FIGS. 13a and 13b are chemical structures of known antilipidemic agentshaving allosteric activity, but which are inactive in the presence ofphysiological levels of serum albumin;

FIGS. 14a-r are chemical structures of several families of allosterichemoglobin modifiers according to this invention;

FIG. 15 is a synthetic pathway for preparing the compounds shown in FIG.14c;

FIG. 16 is a synthetic pathway for preparing the compounds shown in FIG.14d and 14l;

FIG. 17 is a synthetic pathway for preparing the compounds shown in FIG.14e;

FIG. 18 is a synthetic pathway for preparing the compounds shown in FIG.14f;

FIG. 19 is a synthetic pathway for preparing the compounds shown in FIG.14g;

FIG. 20 is a synthetic pathway for preparing the compounds shown in FIG.14h;

FIG. 21 is a synthetic pathway for preparing the compounds shown in FIG.14i;

FIGS. 22a-b are synthetic pathways for preparing the compounds shown inFIGS. 14j and 14q;

FIG. 23 is a synthetic pathway for preparing the compounds shown in FIG.14m;

FIG. 24 is a synthetic pathway for preparing the compounds shown in FIG.14n;

FIG. 25 is a synthetic pathway for preparing the compounds shown in FIG.14o;

FIG. 26 is a synthetic pathway for preparing the compounds shown in FIG.14p; and

FIG. 27 is a synthetic pathway for preparing the compounds shown in FIG.14r.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION

Referring now to the drawings and, more particularly, to FIGS. 1a-cwhich illustrate the general structural formula of particularlypreferred compounds contemplated for use in the present invention andfirst and second subsets of the general structural formula,respectively. With reference to the general structural formula of FIG.1a, the X, Y, and Z moieties may be CH₂, NH, S, SO₂, CO, or O with thecaveat that the X, Y, and Z moieties are each different from oneanother. In addition, R₂₋₆ are either hydrogen, halogen, a substitutedor unsubstituted C₁₋₃ alkyl group (up to three carbons in length), or aC₁₋₃ ester or ether, and these moieties may be the same or different, orthey may be alkyl moieties of aliphatic or aromatic rings incorporatingtwo R₂₋₆ sites. The R₇₋₈ positions are hydrogen, halogen, methyl, ethyl,propyl, isopropyl, neopentyl, butyl, or substituted or unsubstitutedaryl (phenyl, naphthyl, etc.) groups and these moieties may be the sameor different, or they may be alkyl moieties as part of a substituted orunsubstituted aliphatic (e.g., cyclobutyl, cyclopentyl, or cyclohexyl)ring connecting R₇ and R₈. The R₉ position is a hydrogen, C₁₋₃loweralkyl such as methyl, ethyl or propyl, or a salt cation such assodium, potassium, or ammonium, etc.

In the first subset of compounds defined in FIG. 1b, X and Z may each beCH₂, NH, S, SO₂ or O, with the caveat that X and Z are differentconstituents. The first subset of compounds includes, in addition toothers, the following four groupings:

Group I: 2- 4-((aryl)acetamido)phenoxy!-2-methyl propionic acidcompounds having the general structural formula illustrated in FIG. 2C;

Group II: 2- 4-(((aryl)oxy)carbonyl)amino)phenoxy!-2-methyl propionicacid compounds having the general structural formula illustrated in FIG.3;

Group III: 2- 4-((((aryl)amino)carbonyl)methyl)phenoxy!-2-methylpropionic acid compounds having the general structural formulaillustrated in FIGS. 4 and 6a; and

Group IV: 2- 4-(((aryl)amino)carbonyl)oxy)phenoxy!-2-methyl propionicacid compounds having the general structural formula illustrated in FIG.5C.

In the second subset of compounds defined in FIG. 1c, X and Z may eachbe CO, O, S, SO₂ or CH₂, with the caveat that when X and Z are differentconstituents. The second subset of compounds includes, in addition toother, the following two groupings:

Group V: 2- 4-(((aryloyl)amino)methyl)phenoxy!-2-methyl propionic acidcompounds having the general structural formula illustrated in FIG. 7b.

Group VI: 2- 4-((((aryl)methyl)amino)carbonyl)phenoxy!-2-methylpropionicacid compounds which are the subject matter of the co-pending U.S.patent application Ser. No. 07/623,346 to Abraham et al. filed Dec. 7,1990.

The R₂₋₉ substituents in FIGS. 1b-c are the same as those defined withreference to FIG. 1a. The synthesis of specific chemical compoundswithin the first five groups of compounds is provided in the followingexamples with reference to FIGS. 2-7. The synthesis of specific chemicalcompounds in the sixth group is explained in detail in co-pending U.S.patent application Ser. No. 07/623,346 to Abraham which is hereinincorporated by reference. All compounds which were prepared werechecked by thin layer chromatography (TLC) for purity and the structureelucidation was based on NMR and IR spectroscopy and elemental analysis.

EXAMPLE 1

FIG. 2A illustrates a reaction scheme for preparing2-(4-aminophenoxy)-2-methyl propionic acid, a compound that is useful asa precursor in the preparation of Group I compounds. In accordance withthe scheme of FIG. 2A, 8 grams (g) (0.2 mol) of pulverized sodiumhydroxide is added to a suspension of 5.28 g (0.035 mol) ofp-acetamidophenol in 23 ml (0.4 mol) of acetone. The reaction mixture isstirred at room temperature for 1/2 hour. Subsequently, 3.58 g (0.03mol) of chloroform is added dropwise over the course of 30 minutes. Thereaction mixture is stirred overnight at room temperature and acetone isremoved under vacuum. The residue is dissolved in water (10 ml),followed by acidification with 37% hydrochloric acid (HCl) to produce apale yellow precipitate of 2-(4-acetaminophenoxy)-2-methyl propionicacid (5 g, 60% yield), crystallized from methanol, mp 69°-71° C.

¹ HNMR: (CD3OD) δ 7.1 (m,4H) ArH, 2.05 (s,3H), CH₃, 1.45, (s,6H) 2CH₃

1.18 g (0.005 mol) of the 2-(4-acetaminophenoxy)-2-methyl propionic acidis boiled in 10% KOH (60 ml) for 1/2 hour. The solution is then cooledand acidified with acetic acid to yield 0.6 g (62% yield) of2-(4-aminophenoxy)-2-methyl propionic acid as a yellowish white powder,mp 214°-16° C.

¹ H NMR: (DMSOd6+TMS) δ 6.6 (m,4H)ArH, 1.35 (s,6H, 2CH₃)

EXAMPLE 2

FIG. 2B illustrates another reaction scheme for preparing2-(4-aminophenoxy)-2-methyl propionic acid. In accordance with thescheme of FIG. 2B, 8 grams of potassium hydroxide is dissolved in 32 mlof water and the resultant KOH solution is admixed with 280 ml of 3%hydrogen peroxide. 11.3 g (0.058 mol) of 2-(4-cyanophenoxy)-2-methylpropionic acid is slowly added to the KOH/H₂ O₂ solution and thereaction mixture is stirred for about one hour until the exotherm andevolution of gas has ceased. The mixture is then cooled and acidifiedwith concentrated hydrochloric acid. The 2-4-(carboxamido)phenoxy!-2-methyl propionic acid product is obtained as awhite solid (9.8 g, 79% yield). The product is crystallized from ethanolto produce pure white crystals, mp 202°-4° C.

5.57 g (0.025 mol) of the 2- 4-(carboxamido)phenoxy!-2-methyl propionicacid is added gradually with stirring to 100 ml of an ice cold aqueoussolution containing 4.4 g (0,025 mol) of bromine and 11 g (0.25 mol) ofsodium hydroxide. The solution thus obtained is warmed at 75° C. for 1/2hour. After cooling, the solution is acidified with acetic acid givingthe desired 2-(4-aminophenoxy)-2-methyl propionic acid product as 4.0 g(81% yield) of a white precipitate, mp 214°-16° C. The compound isidentical with the product prepared in Example 1.

EXAMPLE 3

FIG. 2C illustrates a general reaction scheme for preparing the Group I2- 4-(arylacetamido)phenoxy!-2-methyl propionic acids. In accordancewith the illustrated scheme, 1 g (0.005 mol) of2-(4-aminophenoxy)-2-methyl propionic acid is dissolved with stirring in10 ml of water containing 0.41 g (0.1 mol) of NaOH. To this solution,0.79 g (0.005 mol) of phenyl acetyl chloride in 5 ml of tetrahydrofuran(THF) is gradually added over a period of about 15 minutes. After theaddition is complete the pH of the reaction mixture should be alkaline(if not a few drops of 2N NaOH is added to assure alkalinity). Thereaction mixture is continuously stirred for 1 hour. Thereafter, the THFis evaporated in vacuo, and the solution is then diluted with 5 ml waterand acidified with concentrated hydrochloric acid. The product isextracted with ethyl ether (2×20 ml), washed with water (3×20 ml), andthen dried over anhydrous MgSO₄. Upon addition of petroleum ether to theether solution, 0.9 g (56% yield) of the 2-4-(phenylacetamido)phenoxy!-2-methyl propionic acid product precipitatesas a pale brown solid, mp 173°-175° C.

¹ H NMR: (DMSOd6) 10 (s,1H, COOH), 7.5-6.7 (m, 9H, ArH), 3.55 (s, 2H,CH₂), 1.4 (s, 6H, 2CH₃) Anal: C₁₈ H₁₉ NO₄ Calculated C 69.00 H 6.07 N4.47 Found C 68.86 H 6.14 N 4.42

EXAMPLE 4

The procedure of Example 3 is followed as above, except that 0.005 molof 4-chlorophenyl acetyl chloride is substituted for the phenyl acetylchloride. In this case the product (57% yield) is 2-4-(p-chlorophenylacetamido)phenoxy!-2-methyl propionic acid, mp 168°-71°C.

¹ H NMR: (DMSOd6) δ 10 (s, 1H, COOH), 7.6-6.7 (m, 8H, ArH), 3.6 (s, 2H,CH₂), 1.4 (s, 6H, 2CH₃) Anal: C₁₈ H₁₈ NO₄ Cl Calculated C 62.15 H 5.17 N4.02 Cl 10.12 Found C 62.16 H 5.25 N 3.98 Cl 10.25

The 4-chlorophenyl acetyl chloride for the foregoing synthesis isprepared by heating to reflux a suspension of 1 g (0.006 mol) of4-chlorophenyl acetic acid in 1.07 g (0.009 mol) of thionyl chloridewith stirring for 1 hour. After cooling, excess thionyl chloride isevaporated under vacuum to present the 4-chlorophenyl acetyl chlorideproduct as a yellow oil (1 g, 83% yield).

EXAMPLE 5

FIG. 3 illustrates a general reaction scheme for preparing the Group II2- 4-(((aryloxy)carbonyl) amino)phenoxy!-2-methyl propionic acids. Inaccordance with the illustrated scheme, a solution consisting of 0.15 g(0.001 mol) of phenyl chloroformate in 3 ml THF is gradually added to anice cold solution containing 0.3 g (0.001 mol) of 2-(4-aminophenoxy)-2-methyl propionic acid and 0.17 g (0.002 mol) of sodiumbicarbonate in 10 ml of water (10 ml). The reaction mixture is stirredfor 1/2 hour at 0° C., followed by stirring for 1 hour at roomtemperature. The THF is removed in vacuo and 10 ml of water is added.Then, the reaction mixture is acidified with concentrated hydrochloricacid and extracted with ethyl ether (2×20 ml). The ether solution iswashed with water (3×20 ml) and dried over anhydrous MgSO₄. The desiredproduct, 2- 4-((((phenyl)oxy)carbonyl)amino)phenoxy!-2-methyl propionicacid, is precipitated from the ether solution by addition of petroleumether as a white solid, 0.15 g (31% yield), mp 183°-5° C.

¹ H NMR: (DMSOd6) δ 10 (s, 1H, COOH), 7.55-6.75 (m, 9H, ArH), 1.4 (s,6H, 2CH₃) Anal: C₁₇ H₁₇ O₅ N Calculated C 64.76 H 5.39 N 4.44 Found C64.65 H 5.45 N 4.43

EXAMPLE 6

The procedure for Example 5 is followed as above except that 0.001 molof 4-chlorophenyl chloroformate is substituted for the phenylchloroformate. In this case the 2-4-((((p-chlorophenyl)oxy)carbonyl)amino)phenoxy!-2-methyl propionic acidproduct is obtained as a white precipitate, 0.15 g (28% yield), mp179°-82° C.

¹ H NMR: (DMSOd₆ +TMS) δ 7.6-6.8 (m, 8H, ArH), 1.4 (s, 6H, 2CH₃) Anal:C₁₇ H₁₆ O₅ NCl Calculated C 58.36 H 4.57 Cl 10.15 Found C 58.16 H 4.68Cl 10.35

EXAMPLE 7

FIG. 4 illustrates a general reaction scheme for preparing the Group IIIcompounds of the invention. In accordance with the illustrated scheme,5.2 g (34 mmol) of 4-hydroxyphenylacetic acid (HPAA) is heated to refluxwith an excess of thionyl chloride (SOCl₂) for 1/2 hour. The reactionmixture is then cooled and excess SOCl₂ is removed under vacuum. Theresidue is reacted for 2 hours with 6.3 g (68 mmol) of aniline in 50 mlof refluxing xylene. The reaction mixture is then cooled, washed withdilute HCl, water and brine and extracted with aqueous 2N NaOH. Thecombined alkali layer is washed with ether, cooled and acidified toprovide 7 g of solid N-phenyl-4-hydroxybenzyl amide (C₁₄ H₁₂ NO₂) as anintermediate product (90% yield), mp 138° C. The intermediate product isrecrystallized from a 1:2 mixture of acetone and petroleum ether and a1.13 g (5 mmol) portion is O-alkylated for 12 hours using the procedureof Example 1 with 20 ml acetone, 2.75 g NaOH and 1.25 ml CHCl₃. Thefinal product is 2- 4-((((phenyl)amino)carbonyl)methyl)phenoxy!-2-methylpropionic acid (C₁₈ H₁₉ NO₄), 1.2 g (76% yield), mp 198° C.

EXAMPLE 8

The procedure of Example 7 is repeated using 8.6 g (68 mmol) of4-chloroaniline rather than the aniline. In this case, the intermediateproduct is N-(4-chlorophenyl)-4-hydroxy benzylamide (C₁₄ H₁₂ ClNO₂), 7.5g (84% yield), mp 163° C. 1.3 g of the intermediate product isO-alkylated to produce 2- 4-((((4-chlorophenyl)amino)carbonyl)methyl)phenoxy!-2-methyl propionic acid (C₁₈ H₁₈ ClNO₄), 0.86 g (50% yield), mp196° C.

EXAMPLE 9

The procedure of Example 7 is repeated using 2.6 g (17 mmol) of the HPAAand using 5.67 g (35 mmol) of 3,4-dichloroaniline rather than aniline.In this case, the intermediate product isN-(3,4-dichlorophenyl-4-hydroxy benzylamide (C₁₄ H₁₁ Cl₂ NO₂). 1.48 g (5mmol) of the intermediate is O-alkylated to produce 2-4-(((3,4-dichlorophenyl)amino)carbonyl)methyl)phenoxy!-2-methylpropionic acid (C₁₈ H₁₇ Cl₂ NO₄), 0.76 g (40% yield), mp 174° C.

EXAMPLE 10

The procedure of Example 7 is repeated using 2.6 (17 mmol) of the HPAAand using 5.7 g (35 mmol) of 3,5-dichloroaniline rather than aniline. Inthis case, the intermediate product is N-(3,5-dichlorophenyl-4-hydroxybenzylamide (C₁₄ H₁₁ Cl₂ NO₂). 1.48 g (5 mmol) of the intermediate isO-alkylated to produce 2-4-((((3,5-dichlorophenyl)amino)carbonyl)methyl)phenoxy!-2-methylpropionic acid (C₁₈ H₁₇ Cl₂ NO₄), 0.8 g (42% yield), mp 138° C.

EXAMPLE 11

The procedure of Example 7 is repeated using 0.95 g (6 mmol) of theHPAA, 2.6 g (12 mmol) of 3,4,5-trichloroaniline rather than aniline, and25 ml of refluxing xylene. In this case, the intermediate product isN-(3,4,5-trichlorophenyl)-4-hydroxy benzylamide. 0.50 g (1.5 mmol) ofthe intermediate product is O-alkylated using 10 ml acetone, 0.82 g NaOHand 0.37 ml CHCl₃ to produce 2-4-((((3,4,5-trichlorophenyl)amino)carbonyl)methyl)phenoxy!-2-methylpropionic acid (C₁₈ H₁₆ Cl₃ NO₄), 0.27 g (43% yield), mp 160° C.

EXAMPLE 12

The procedure of Example 7 is repeated using 5.04 g (32 mmol) of theHPAA, 6 ml (64 mmol) of 4-fluoroaniline rather than aniline, and 25 mlof refluxing xylene. In this case, the intermediate product isN-(4-fluorophenyl)-4-hydroxybenzylamide. 1.22 g (5 mmol) of theintermediate product is O-alkylated to produce 2-4-((((4-fluorophenyl)amino)carbonyl)methyl)phenoxy!-2-methyl propionicacid (C₁₈ H₁₈ FNO₄), 0.74 g (45% yield), mp 198° C.

EXAMPLE 13

The procedure of Example 7 is repeated using 5.04 (32 mmol) of the HPAA,8.05 ml (64 mmol) of 4-trifluoromethylaniline rather than aniline, and25 ml of refluxing xylene. In this case, the intermediate product isN-(4-trifluoromethylphenyl)-4-hydroxy benzylamide. 1.5 g (5 mmol) of theintermediate is used to produce 2-4-((((4-trifluoromethylphenyl)amino)carbonyl)methyl)phenoxy!-2-methylpropionic acid (C₁₉ H₁₈ F₃ NO₄), 0.85 g (44% yield), mp 197° C.

EXAMPLE 14

The procedure of Example 7 is repeated using 5.04 (32 mmol) of the HPAA,8 g (65 mmol) of 4-methyl aniline rather than aniline, and using 25 mlof refluxing xylene. In this case the intermediate product isN-(4-methylphenyl)-4-hydroxy benzylamide. 1.2 g (5 mmol) of theintermediate is used to produce 2-4-((((4-methylphenyl)amino)carbonyl)methyl)phenoxy!-2-methyl propionicacid (C₁₉ H₂₁ NO₄), 0.98 g (65% yield), mp 164° C.

EXAMPLE 15

The procedure of Example 7 is repeated using 3.26 (21 mmol) of the HPAA,5.3 ml (42 mmol) of 3,5-dimethyl aniline rather than aniline, and 25 mlof refluxing xylene. In this case the intermediate product isN-(3,5-dimethylphenyl)-4-hydroxy benzylamide. 1.27 g (5 mmol) of theintermediate is used to produce 2-4-((((3,5-dimethylphenyl)amino)carbonyl)methyl)phenoxy!-2-methylpropionic acid (C₂₀ H₂₃ NO₄), 1.15 g (68% yield), mp 85° C.Alternatively, the procedure outlined in the German Patent Application2,432,560, which is herein incorporated by reference, can be followed toproduce the compound of this Example 15.

EXAMPLE 16

The procedure of Example 7 is repeated using 5.04 (32 mmol) of the HPAA,10 ml (64 mmol) of 4-isopropyl aniline rather than aniline, and using 25ml of refluxing xylene. In this case the intermediate product isN-(4-isopropylphenyl)-4-hydroxybenzylamide. 1.34 g (5 mmol) of thesemi-solid, thick viscous liquid intermediate is used to prepare 2-4-((((4-isopropylphenyl)amino)carbonyl)methyl)phenoxy!-2-methylpropionic acid (C₂₁ H₂₅ NO₄), 1.1 g (61% yield), mp 141° C.

EXAMPLE 17

With reference to FIGS. 5A, 5B and 5C, a scheme is illustrated forpreparing Group IV compounds. In accordance with FIG. 5A, aniline oraniline derivatives may be reacted with phosgene to obtain the carbamoylchloride. In accordance with FIG. 5B, hydroquinone may be monoacetylatedusing acetic anhydride. The product is then O-alkylated using acetone,CHCl₃ and KOH and then hydrolyzed using a base. The products of thereactions of FIGS. 5A and 5B may then be reacted according to thereaction scheme of FIG. 5C to produce the Group IV 2-4-(((arylamino)carbonyl)oxy)phenoxy)!-2-methyl propionic acids.

EXAMPLE 18

As an alternative to the reaction scheme described in Example 7 andshown in FIG. 4, the Group III compounds may be prepared according tothe scheme shown in FIG. 6a. 5.2 g (32 mmol) of HPAA, 6.3 g (68 mmol) ofaniline, and 25 ml of mesitylene are heated to reflux. 0.74 g (8mmol) ofphosphorous pentachloride is added to the refluxing mixture and thereflux is continued for an additional two hours. The reaction mixture issubsequently cooled, washed with dilute HCl, water and brine, andextracted with aqueous 2N sodium hydroxide NaOH. The combined alkalilayer is washed with ether, cooled and acidified to provide 7 g (90%yield) of solid N-phenyl-4-hydroxybenzyl amide (C₁₄ H₁₂ NO₂) as anintermediate product, mp 138° . The intermediate product isrecrystallized from a 1:2 mixture of acetone:petroleum ether and a 1.13g (5 mmol) portion is O-alkylated. 1.6 g (30 mmol) of pulverized sodiumhydroxide is added to a solution of N-phenyl-4-hydroxybenzamide (1.13 g,5 mmol) in 20 ml of acetone. The reaction mixture is stirred overnightat room temperature and acetone is removed under vacuum. The residue isdissolved in 10 ml of water and acidified with 2N HCl to produce a paleyellow solid. The solid is separated, dissolved in methanol,charcoalated, and solvent evaporated to provide 2-4-((((phenyl)amino)carbonyl)methyl)phenoxy!-2-methyl propionic acid (C₁₈H₁₉ NO₄), 1.2 g (76% yield), mp 198° C. The last step in the procedureshown in FIG. 6a is the conversion of the acid to the sodium salt viaits reaction with sodium bicarbonate. Similar reactions with other saltcations such as potassium and ammonium or reactions to form esters(e.g., methyl, ethyl, propyl, etc.) can also be performed.

EXAMPLE 19a

FIG. 6b presents a similar reaction scheme to FIG. 6a, except that 3-rather than 4-hydroxyphenylacetic acid (HPAA) is used as the precursormaterial so that the final compound has a meta rather than a parasubstitution. In addition, rather than reacting with acetone (dimethylketone) a diethyl ketone is used to position ethyl, rather than methyl,moieties in the group substituted on one of the phenyl rings. Byexample, 1.5g (10mmol) 3-HPAA and 2.6g (20 mmol) 4-chloroaniline in 20ml of mesitylene was heated to reflux. Then 0.33 g (2.55 mmol) PCl₅ wasthen slowly added to the above refluxing solution and the refluxing wascontinued for two hours. The reaction mixture was then cooled and thenworked up as described above to yield 2.2 g (90% yield) of 3-((4-chloroanilino) carbonyl)methyl!phenol. As described above,chloroform (0.8 ml) was added to a stirred and ice-cooled mixture of1.23 grams of 3- ((4-chloroanilino)carbonyl)methyl!phenol and 1.6 g NaOHin 15 ml of acetone. The reaction mixture was allowed to warm to roomtemperature and stirring continued for an additional 10 hours. The usualwork-up yielded 2-3-(((4-chloroanalino)carbonyl)methyl)!phenoxy!-2-methylpropionic acid asa low temperature melting sticky solid (C,H,Cl,N analysis yielded (C₁₈H₁₈ ClNO₄); NMR δPPM: 1.42 (6H, s, CH₃), 3.61 (2H, s, benzylic CH₂), and6.6-7.75 (8H, m, aromatic H)). However, rather than using acetone as thereaction solvent, diethylketone can be used in the same manner asdescribed above to yield the butanoic acid (as opposed to propanoicacid) structure shown in FIG. 6b.

EXAMPLE 19b

FIG. 6c describes a general reaction scheme to introduce groups largerthan methyl on the acid moiety of compounds of Group III.

N-(3,5-dimethylphenyl)-4-hydroxyphenylacetamide (3.06 gms, 12 mmol) inTHF is treated with 2.4 gms (60 mmol) of NaOH at -20° C. 5.45 ml (60mmol) of isobutyraldehyde and 4.8 ml (60 mmol) of CHCl₃ is addeddropwise simultaneously at -20° C. and stirring continued overnight atroom temperature. THF is removed under vacuum and the residue isdissolved in water, followed by acidification with 35% HCl. Theprecipitated solid is extracted into ether and treated with 6% NaHCO₃solution. The aqueous layer on acidification yields the product3-methyl-2-4-((((3,5-dimethylphenyl)amino)carbonyl)methyl)phenoxy!butanoic acid(C₂₁ H₂₅ NO₄) (yield 1 gm, 23.5%) mp (uncorrected) 96°-100° C.

NMR: acetone-d₆ 1.1 (6H, d, 2 CH₃ of isopropyl), 1.3 (1H, m, C_(H) ofisopropyl), 6.7-7.3 (7H, m, ArH)

EXAMPLE 19c

The procedure of Example 19b is repeated using 0.507 gins (2 mmol) ofN-(3,5-dimethylphenyl)-4-hydroxyphenyl acetamide, 0.8 gms (20 mmol) ofNaOH, 1.6 ml (20 mmol) of CHCl₃ and 2 ml (20 mmol) of3,3-dimethylphenyl)amino)carbonyl)methyl)phenoxy!pentanoic acid (C₂₃ H₂₉NO₄) (yield 0.310 gms, 40.6%) mp (uncorrected) 118°-125° C.

NMR: acetone-d₆, 1.0 (9H, s, CH₃), 1.8 (m, CH₂), 2.2 (6H, s, 2ArCH₃),3.6 (2H, s, CH₂ adjacent to carbonyl), 4.6 (1H, dd, CH), 6.7-7.3 (7H, m,ArH).

EXAMPLE 20

With reference to FIGS. 7a, a general reaction scheme for preparing 2-4-(aminomethyl)phenoxy!-2-methyl propionic acid, a compound that isuseful as a precursor to the preparation of the Group V compounds, ispresented. In accordance with the illustrated scheme, 2-4-cyanophenoxy!-2-methyl propionic acid (2g, 9mmol), prepared asdescribed in Example 2, and 75 ml of ethanol were placed in a 250 mlParr hydrogenation bottle. The solution was acidified with concentratedhydrochloric acid (3 ml), then 10% palladium on activated charcoal (0.2g, 10% wt) was added to the mixture. The reaction mixture was placed ona Parr hydrogenator apparatus at 45 psi of hydrogen pressure and shakenfor a period of two hours. The mixture was filtered to remove thecatalyst, and the filtrate concentrated under vacuum. Addition of etherprecipitated hydrochloride salt of the desired product as white, shinycrystals (2.1 g, 87%).

EXAMPLE 21

FIG. 7B illustrates a general reaction scheme for preparing the Group Vcompounds used in the present invention. In accordance with theillustration, a solution of benzoyl chloride (0.14 g, 1 mmol) in THF (3ml) was added over a 15 minute period to a stirred solution of 2-4-(aminomethyl)phenoxy!-2-methylpropionic acid (0.24 g, 1 mmol) and NaOH(0.08 g, 2 mmol) in 10 ml of water. After the addition of the benzoylchloride was completed, the reaction mixture was stirred for 1 hour atroom temperature. THF was evaporated in vacuo. Acidification of theresidue provided the desired compound as an oil which was extracted withether. The organic layer was washed with water, brine, and aired overanhydrous MgSO₄. Subsequent addition of petroleum ether precipitated 2-4-(benzoylamino)methyl)phenoxy!-2-methyl propionic acid (C₁₈ H₁₉ NO₄) asa white solid (0.15 g, 48%) mp 176°-179° C.

NMR: (DMSO-d₆) δ 1.45 (6H, s, 2CH₃), 4.4 (2H, d, CH₂), 6.8-7.2 (4H, dd,J=9 Hz, aromatic H, 7.4-8 (5 H, m, aromatic H), 9, (1 H, br t, NH).

EXAMPLE 22

The procedure of Example 21 is repeated using 2-chlorobenzoyl chloride(1 mmol) rather than benzoyl chloride. In this case, the product (58%yield) is 2 4-(((2-chlorobenzoyl)amino)methyl)phenoxy!-2-methylpropionic acid (C₁₈ H₁₈ ClNO₄) mp 135°-137° C.

EXAMPLE 23

The procedure of Example 21 is repeated, except that 1 mmol of3-chlorobenzoyl chloride is substituted for benzoyl chloride. In thiscase, the product (53% yield) is 2-4-(((3-chlorobenzoyl)amino)methyl)phenoxy!-2-methyl propionic acid (C₁₈H₁₈ ClNO₄) mp 145°-146° C.

EXAMPLE 24

The procedure of Example 21 is repeated, except that 1 mmol of4-chlorobenzoyl chloride is substituted for benzoyl chloride. In thiscase, the product (63% yield) is 2-4-(((4-chlorobenzoyl)amino)methyl)phenoxy!-2-methyl propionic acid (C₁₈H₁₈ ClNO₄) mp 186°-189° C.

EXAMPLE 25

The procedure of Example 21 is repeated, except that 1 mmol of3,4-dichlorobenzoyl chloride is substituted for benzoyl chloride. Inthis case, the product (57% yield) is 2-4-(((3,4-dichlorobenzoyl)amino)methyl)phenoxy!-2-methyl propionic acid(C₁₈ H₁₇ Cl₂ NO₄) mp 186°-189° C.

EXAMPLE 26

The procedure of Example 20 is repeated, except that 1 mmol of3,5-dichlorobenzoyl chloride is substituted for benzoyl chloride. Inthis case, the product (43% yield) is 2-4-(((3,5-dichlorobenzoyl)amino)methyl)phenoxy!-2-methyl propionic acid(C₁₈ H₁₇ Cl₂ NO₄) mp 110°-113° C.

EXAMPLE 27

The procedure of Example 20 is repeated, except that 1 mmol of3,4,5-trichlorobenzoyl chloride is substituted for benzoyl chloride. Inthis case, the product is 2-4-(((3,4,5-trichlorobenzoyl)amino)methyl)phenoxy!-2-methyl propionicacid (C₁₈ H₁₆ Cl₃ NO₄) mp 151°-152° C.

EXAMPLE 28

The procedure of Example 21 is repeated, except that 1 mmol of p-toluenesulphonyl chloride is substituted for benzoyl chloride. In this case,the product is 2- (4-methyl phenyl)sulfonamide methyl)phenoxy-2-methylpropionic acid (C₁₈ H₂₁ NSO₅) mp 108° C.

EXAMPLE 29

N-(3,5-dimethylphenyl)-4-hydroxyphenylacetamide (3.06 gms, 12 mmol) inTHF is treated with 2.4 gms (60 mmol) of NaOH at -20° C. Subsequently5.889 gms (60 mmol) of cyclohexanone and 4.8 ml (60 mmol) of CHCl₃ isadded dropwise simultaneously at -20° C. and stirred overnight at roomtemperature. THF is removed under vacuum and the residue is dissolved inwater, followed by acidification with 35% HCl. The precipitated solid isextracted into ether and treated with 6% sodium bicarbonate solution.The aqueous layer on acidification with HCl yields the product 1-4-(((3,5-dimethylphenyl)amino)carbonyl)methyl)phenoxy!cyclohexanecarboxylicacid (C₂₃ H₂₇ NO₄). The product was purified by repeated extraction intoether and NaHCO₃.

NMR: (acetone-d₆) 2.2 (6H, s, 2CH₃), 3.6 (2H, s, CH₂), 1.5-2 (brm,cyclohexyl ring protons), 6.7-7.2 (7H, m, ArH).

EXAMPLE 30

The procedure of Example 29 is repeated with 1.02 gms (4 mmol) ofN-(3,5-dimethylphenyl)-4-hydroxyphenyl acetamide, 0.8 gms (20 mmol) ofNaOH, 1.6 mL (20 mmol) of CHCl₃ and 1.77 mL (20 mmol) of cyclopentanoneto prepare 1- 4-((((3,5-dimethylphenyl)amino)carbonyl)methyl)phenoxy!cyclopentane carboxylic acid (C₂₂ H₂₅ NO₄) (yield 0.100 gms,6.8%) mp (uncorrected) 88°-94° C.

NMR: (acetone-d₆) 1.8-2.3 (brm, cyclopentyl ring protons), 2.2 (6H, s, 2CH₃), 3.56 (2H, s, CH₂), 6.7-7.3 (7H, m, ArH)

EXAMPLE 31

The procedure of Example 29 is repeated with 1.5 gms (5 mmol) ofN-(3,5-dichlorophenyl)-4-hydroxyphenyl acetamide, 1 gm (25 mmol) ofNaOH, 2 ml (25 mmol) of CHCl₃ and 1.17 gms (12 mmol) of cyclohexanone toprepare 1-4-((((3,5-dichlorophenyl)amino)carbonyl)methyl)phenoxy!cyclohexanecarboxylicacid (C₂₀ H₁₉ NO₄ Cl₂) (yield 0.22 gms, 10.3%) mp (uncorrected) 80°-90°C.

NMR: (acetone-d₆) 1.5-2 (10H, brm, cyclohexyl ring protons), 3.6 (2H, s,CH₂), 6.8-7.8 (7H, m, ArH).

EXAMPLE 32

The procedure of Example 30 is repeated usingN-(3,5-dichlorophenyl)-4-hydroxyphenylacetamide and cyclopentanone toprepare 1-4-((((3,5-dichlorophenyl)amino)carbonyl)methyl)phenoxy!cyclopentanecarboxylicacid (C₁₉ H₁₇ NO₄ Cl₂).

Examples 1 through 32 outline the synthesis procedures for producingseveral compounds within the family of compounds defined by the generalstructural formula of FIG. 1a. Specifically, Examples 1-19 disclosesynthesis procedures for Groups 1-4 compounds within the subset definedby the structural formula of FIG. 1b and Examples 20-27 disclosesynthesis procedures for Group 5 compounds within the subset defined bythe structural formula of FIG. 1c. The co-pending U.S. patentapplication Ser. No. 07/623,346 to Abraham et al. filed Dec. 7, 1990,describes the synthesis procedures for Group 6 compounds within thesubset defined by the structural formula of FIG. 1c. Example 28 providessynthesis procedures where a sulfur or sulfur dioxide is present at theX, Y, or Z positions with respect to FIG. 1a. Examples 29-32 providesynthesis procedures where a five or six membered aliphatic ring isconnected to the propionic acid tail portion of the FIG. 1a molecule. Itshould be understood that many other compounds within the family ofcompounds used in the present invention can easily be synthesized bychanging the starting materials. All compounds within the family wouldhave a similar mode of binding and would, therefore, all should have theeffect of shifting the allosteric equilibrium of hemoglobin towardsfavoring the low affinity "T" state.

One broad family of compounds contemplated for use in this inventionincludes compounds defined by the formula: ##STR2## where R₂ is asubstituted or unsubstituted aromatic such as a phenyl, naphthyl, orindanyl, or hetrocyclic aromatic, or a substituted or unsubstitutedalkyl ring compound, such as a cyclohexyl or adamantyl, or a substitutedor unsubstituted phthalimide compound that incorporates X and Y where Xis a carbonyl, Y is a nitrogen and R₂ completes the phthalimide compoundby being bonded to both X and Y, and, if R₂ is not a phthalamidecompound, where X, Y, and Z are CH₂, NH, S, SO₂, CO or O with the caveatthat the X, Y, and Z moieties are each different from one another, andwhere R₁ has the formula: ##STR3## where R1 can be connected to anyposition on the phenyl ring and R₃ and R₄ are hydrogen, halogen, methyl,ethyl, propyl, isopropyl, neopentyl, butyl, or substituted orunsubstituted aryl (phenyl, naphthyl, etc.) groups and these moietiesmay be the same or different, or alkyl moieties as part of an aliphaticring connecting R₃ and R₄, such as substituted 3, 4, 5, and 6 carbonatom rings, and R₅ is a hydrogen, C₁₋₃ loweralkyl such as methyl, ethylor propyl, or a salt cation such as sodium, potassium, or ammonium. Tothis end, compounds having a naphthyl, adamantyl, or indanyl group at R₁instead of the substituted phenyl like that shown in FIG. 1a have beenprepared using substantially the same synthetic routes as describedabove. In addition, compounds having a phthalimide-like structure havealso been synthesized as shown in FIGS. 12a-b and described below inEXAMPLE 33.

EXAMPLE 33

FIGS. 12a and 12b show alternative synthesis routes for preparing 2-4-((phthalamido)N-methyl)phenoxy)-2-methyl proprionic acid. Phthalicanhydride (0.75 g; 5 mmol) and 2 4-((amino)methyl) phenoxy!-2-methylproprionic acid (see FIG. 2B) were refluxed in 25 ml of toluene in thepresence of 1 ml triethylamine. Water was removed azeotropically. Afterfour hours of refluxing, the reaction mixture was cooled, toluene wasseparated, and the above-described work up was provide to yield acrystalline white residue (90% yield; mp 149° C.; NMR δ ppm: 1.46(6H, s,CH₂), 4.65(2H, s, CH₂), 6.75 and 7.2 (4H, d, J=6 Hz, aromatic H of aparasubstituted ring), and 7.85 (4H, s, aromatic H of a phthalimideunit).

To test the compounds of the invention for physiological activity, humanblood was obtained from the Central Blood Bank, Richmond, Va. Theextraction, chromatography, and characterization of isolated hemoglobinmethods used by the inventors were identical to those described by Dozyand Huisman in J. of Chromatography, Vol 32, (1968) pp. 723 and in TheChromatography of Hemoglobin, H. J. Schroeder and D. H. J. Huisman, Ed.Marcel Dekker Inc. N.Y. (1980) which are herein incorporated byreference. The purity of normal hemoglobin (HbA) was determined by gelelectrophoresis, using a Gelman semimicroelectrophoresis chamber. Theconcentration of hemoglobin was determined according to thecyanmethemoglobin method described in Zijlstra, Clin. Chem. Acta., Vol5, pp. 719-726 (1960), and Zijlstra and Van Kamper, J. Clin. Chem. Clin.Biochem., Vol. 19, p. 521 (1981) which are herein incorporated byreference. All purified hemoglobin solutions were stored in liquidnitrogen. The reagents and buffers were purchased from the followingsources: Fischer Scientific, Sigma Chemical Company, and Pharmacia andResearch Chemicals, Inc.

Oxygen equilibrium curves were determined on an AMINCO™ HEM--O--SCANoxygen dissociation analyzer available from Travenol Laboratories. HbAwas prepared as follows: 20 ml of whole blood from a nonsmoking donor(blood bank, Richmond, Va.) was drawn into a heparinized vacutainer. Theblood was immediately packed in ice (to prevent MetHb formation) andthen centrifuged (10 minutes at 2500 rpm) to separate the plasma andbuffy coat from the packed erythrocytes. After centrifugation wascompleted, the plasma and buffy coat were removed by aspiration and thecells washed three times with 0.9% NaCl containing 40 mg ofethylenediamine-tetraacetic acid (EDTA) per liter and then once with1.0% NaCl containing 40 mg of EDTA/L. The cells were lysed by theaddition of one to two volumes of deionized water containing 40 mg ofEDTA/L. The mixture was allowed to stand for 30 minutes with occasionalmixing before being centrifuged for two hours at 10,000 rpms at 4° C.for two hours to remove the remaining cell stroma. The supernatant wasfurther purified by either gel filtration with Sephadex G-25 or dialysisagainst pH 8.6 tris buffer (50 mM, containing 40 mg. of EDTA/L). Thesodium chloride free hemoglobin solution was chromatographed onDEAE-Sephacel ion-exchange resin (Sigma) preequilibrated with Trisbuffer (pH 8.6, 50 mM, containing 40 mg of EDTA/L), the HbA fraction wasthen eluted with pH 8.4 Tris buffer. The pure HbA fraction (identifiedby electrophoresis) was concentrated using a Schleicher and Schuellcollodion bag apparatus (Schleicher and Schuell, Inc.) with HEPES buffer(150 mM, pH 7.4) as the exchange buffer. The hemoglobin concentrationwas then determined using the above-noted cyanomethemoglobin method. Thehemoglobin concentration at this point was usually found to be around 35g% or approximately 5.5 mM. Less than 5% methemoglobin was noted evenafter several days at 4° C.

All compounds were mixed with one equivalent of sodium bicarbonate(NaHCO₃) (this process converts the carboxylic acid moiety to a sodiumsalt; see FIG. 6a; it is noted that other salts can be formed by similarprocess), then dissolved in the HEPES buffer to give 20 mM solutions.Just prior to running the oxygen equilibrium curve, the hemoglobin andthe drug were mixed in a 1:1 ratio (50 μl of hemoglobin plus 50 μl ofdrug) to give 2.75 mM hemoglobin with a drug concentration of 10 mM. Thecontrol was prepared by the addition of 50 μl of hemoglobin to 50 μl ofthe HEPES buffer.

Table 1 presents the measured P₅₀ value, the P₅₀ control value, and theratio of the measured P₅₀ value to the control (P₅₀ /P₅₀ c) for normalhemoglobin treated with several synthesized compounds. Note that the X,Y, Z and R₂ -R₈ positions relate to FIG. 1a.

                                      TABLE 1                                     __________________________________________________________________________    R.sub.2                                                                         R.sub.3                                                                          R.sub.4                                                                          R.sub.5                                                                          R.sub.6                                                                         X  Y  Z   R.sub.7                                                                          R.sub.8                                                                             P.sub.50 c                                                                       P.sub.50                                                                         P.sub.50 /P.sub.50 c                    __________________________________________________________________________    H H  H  H  H CO NH CH.sub.2                                                                          CH.sub.3                                                                         CH.sub.3                                                                            18 35 1.94                                    Cl                                                                              H  H  H  H CO NH CH.sub.2                                                                          CH.sub.3                                                                         CH.sub.3                                                                            18 27.5                                                                             1.52                                    H Cl H  H  H CO NH CH.sub.2                                                                          CH.sub.3                                                                         CH.sub.3                                                                            18 37.5                                                                             2.08                                    H H  Cl H  H CO NH CH.sub.2                                                                          CH.sub.3                                                                         CH.sub.3                                                                            19 48 2.52                                    H Cl Cl H  H CO NH CH.sub.2                                                                          CH.sub.3                                                                         CH.sub.3                                                                            18 40.5                                                                             2.25                                    H Cl H  Cl H CO NH CH.sub.2                                                                          CH.sub.3                                                                         CH.sub.3                                                                            18 47 2.6                                     H Cl Cl Cl H CO NH CH.sub.2                                                                          CH.sub.3                                                                         CH.sub.3                                                                            19 40 2.1                                     H H  H  H  H CH.sub.2                                                                         CO NH  CH.sub.3                                                                         CH.sub.3                                                                            19 35 1.73                                    H Cl H  H  H CH.sub.2                                                                         CO NH  CH.sub.3                                                                         CH.sub.3                                                                            18 44 2.44                                    H H  Cl H  H CH.sub.2                                                                         CO NH  CH.sub.3                                                                         CH.sub.3                                                                            19 44 2.31                                    H H  F  H  H CH.sub.2                                                                         CO NH  CH.sub.3                                                                         CH.sub.3                                                                            18 35 1.94                                    H H  CH.sub.3                                                                         H  H CH.sub.2                                                                         CO NH  CH.sub.3                                                                         CH.sub.3                                                                            18 45 2.5                                     H H  CF.sub.3                                                                         H  H CH.sub.2                                                                         CO NH  CH.sub.3                                                                         CH.sub.3                                                                            18 42 2.33                                    H H  OMe                                                                              H  H CH.sub.2                                                                         CO NH  CH.sub.3                                                                         CH.sub.3                                                                            18 38 2.11                                    H H  Cl Cl H CH.sub.2                                                                         CO NH  CH.sub.3                                                                         CH.sub.3                                                                            18 50 2.77                                    H Me H  Me H CH.sub.2                                                                         CO NH  CH.sub.3                                                                         CH.sub.3                                                                            18 52 2.88                                    H H  H  H  H O  CO NH  CH.sub.3                                                                         CH.sub.3                                                                            18 34 1.88                                    H H  Cl H  H O  CO NH  CH.sub.3                                                                         CH.sub.3                                                                            19 34 1.78                                    H H  H  H  H NH CO CH.sub.2                                                                          CH.sub.3                                                                         CH.sub.3                                                                            19 54 2.84                                    H H  Cl H  H NH CO CH.sub.2                                                                          CH.sub.3                                                                         CH.sub.3                                                                            19 54 2.84                                    H Cl Cl H  H NH CO CH.sub.2                                                                          CH.sub.3                                                                         CH.sub.3                                                                            18 65 3.61                                    H Cl H  Cl H NH CO CH.sub.2                                                                          CH.sub.3                                                                         CH.sub.3                                                                            19 83 4.36                                    H Cl Cl Cl H NH CO CH.sub.2                                                                          CH.sub.3                                                                         CH.sub.3                                                                            19 63 3.3                                     H H  F  H  H NH CO CH.sub.2                                                                          CH.sub.3                                                                         CH.sub.3                                                                            18 45 2.5                                     H H  CF.sub.3                                                                         H  H NH CO CH.sub.2                                                                          CH.sub.3                                                                         CH.sub.3                                                                            18 44 2.44                                    H H  CH.sub.3                                                                         H  H NH CO CH.sub.2                                                                          CH.sub.3                                                                         CH.sub.3                                                                            18 49 2.72                                    H CH.sub.3                                                                         H  CH.sub.3                                                                         H NH CO CH.sub.2                                                                          CH.sub.3                                                                         CH.sub.3                                                                            19 75 3.94                                    Cl                                                                              H  H  H  Cl                                                                              NH CO CH.sub.2                                                                          CH.sub.3                                                                         CH.sub.3                                                                            18 34 1.89                                    H CH.sub.3                                                                         CH.sub.3                                                                         H  H NH CO CH.sub.2                                                                          CH.sub.3                                                                         CH.sub.3                                                                            18 62 3.41                                    NAPHTHYL     NH CO CH.sub.2                                                                          CH.sub.3                                                                         CH.sub.3                                                                            18 58 3.2                                     H Cl H  H  H NH CO CH.sub.2                                                                          CH.sub.3                                                                         CH.sub.3                                                                            18 61 3.4                                     H H  Cl H  H CH.sub.2                                                                         NH CO  CH.sub.3                                                                         CH.sub.3                                                                            18 27 1.5                                     H H  CH.sub.3                                                                         H  H CH.sub.2                                                                         NH CO  CH.sub.3                                                                         CH.sub.3                                                                            19 28 1.47                                    H Cl Cl H  H CH.sub.2                                                                         NH CO  CH.sub.3                                                                         CH.sub.3                                                                            18 28 1.56                                    H H  H  H  H CH.sub.2                                                                         NH CO  CH.sub.3                                                                         CH.sub.3                                                                            19 22 1.16                                    INDANYL      NH CO CH.sub.2                                                                          CH.sub.3                                                                         CH.sub.3                                                                            18 64 3.56                                    ADAMANTYL    NH CO CH.sub.2                                                                          CH.sub.3                                                                         CH.sub.3                                                                            18 32 1.78                                    H CH.sub.3                                                                         H  CH.sub.3                                                                         H NH CO CH.sub.2                                                                          H  CH(CH.sub.3).sub.2                                                                  19 42 2.21                                    H CH.sub.3                                                                         H  CH.sub.3                                                                         H NH CO CH.sub.2                                                                          H  CH.sub.2 C(CH.sub.3).sub.2                                                          19 38 2.0                                     H CH.sub.3                                                                         H  CH.sub.3                                                                         H NH CO CH.sub.2                                                                         cyclopentyl                                                                             19 61 3.2                                     H CH.sub.3                                                                         H  CH.sub.3                                                                         H NH CO CH.sub.2                                                                         cyclohexyl                                                                              19 43 2.26                                    H CH.sub.3                                                                         H  CH.sub.3                                                                         H NH CO CH.sub.2                                                                          C.sub.2 H.sub.5                                                                  C.sub.2 H.sub.5                                                                     19 31 1.63                                    H Cl H  Cl H NH CO CH.sub.2                                                                         cyclohexyl                                                                              19 36 1.89                                    H Cl H  Cl H NH CO CH.sub.2                                                                          C.sub.2 H.sub.5                                                                  C.sub.2 H.sub.5                                                                     18 26 1.44                                    H H  CH.sub.3                                                                         H  H SO.sub.2                                                                         NH CH.sub.2                                                                          CH.sub.3                                                                         CH.sub.3                                                                            18 35 1.94                                    H H  Cl H  H CO NH (CH.sub.2).sub.2                                                                  CH.sub.3                                                                         CH.sub.3                                                                            18 35 1.83(BZF)                               __________________________________________________________________________

It is noted that the P₅₀ control value is less than the P₅₀ for normalhemoglobin under physiological conditions (e.g., 26.5) because here theP₅₀ was made on hemoglobin in solution (outside the red blood cells).Each hemoglobin sample treated with one of the compounds falling withinthe family defined by this invention had a P₅₀ drug value which wasgreater than the P₅₀ control. This response indicates that theallosteric equilibrium for hemoglobin has been shifted towards favoringthe low oxygen affinity "T" state of hemoglobin due to the presence ofthe compounds.

At the bottom of Table 1, the P₅₀ results are presented for bezafibrate(BZF), a known "right-shifting" allosteric hemoglobin modifier. As withall the newly discovered "right-shifting" allosteric hemoglobinmodifiers, the hemoglobin treated with BZF had a higher P₅₀ than the P₅₀for the control.

Table 1 shows the varying R₂₋₆ moieties for the substituted phenylcompounds tested, and when a compound which did not have a substitutedphenyl, the name of the compound is written across R₂₋₆ (e.g., naphthyl,adamantyl, indanyl). The R₇₋₈ moieties were methyl groups for most ofthe compounds tested; however, data for hydrogen, ethyl, isopropyl,isobutyl, cyclopentyl, and cyclohexyl is also presented in Table 1.Table 1 demonstrates the R₇₋₈ moieties may be the same or different andmay be hydrogen, halogen, methyl, ethyl, propyl, isopropyl, neopentyl,butyl, substituted or unsubstituted aryl (phenyl, naphthyl, etc.), oralkyl moieties as part of a substituted or unsubstituted aliphatic ring(cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl) connecting R₇ andR₈. The R₉ moiety was a sodium cation for each compound tested which isderived from the NaHCO₃ treatment prior to testing; however, other saltcations such as ammonia, etc., or esters, ethers, or other derivativescan easily be made within the practice of the invention. Because othercompounds within the family would have a similar mode of binding (e.g,those with different R₂₋₉ moieties), their effect on the P₅₀ value canbe expected to be the same. The phthalimide structure defined by FIGS.12a-b had a mean P₅₀ value (e.g., P₅₀ Drug/P₅₀ Control) of 1.08indicating the allosteric equilibrium for hemoglobin had been shiftedtowards favoring the low oxygen affinity "T" state of hemoglobin by thephthalimide compound.

Table 2 shows the effect some of the compounds on the oxygendissociation of normal hemoglobin in intact human red blood cells(RBCs).

                  TABLE 2                                                         ______________________________________                                        R.sub.2                                                                            R.sub.3                                                                              R.sub.4                                                                             R.sub.5                                                                            R.sub.6                                                                           X    Y   Z    R.sub.7                                                                            R.sub.8                                                                            P.sub.50                   ______________________________________                                        control                        27                                             H    CH.sub.3                                                                             H     CH.sub.3                                                                           H   NH   CO  CH.sub.2                                                                           CH.sub.3                                                                           CH.sub.3                                                                           83                         H    Cl     H     Cl   H   NH   CO  CH.sub.2                                                                           CH.sub.3                                                                           CH.sub.3                                                                           87                         H    CH.sub.3                                                                             H     CH.sub.3                                                                           H   NH   CO  CH.sub.2                                                                           CH.sub.3                                                                           CH.sub.3                                                                           76                         H    Cl     H     Cl   H   NH   CO  CH.sub.2                                                                           CH.sub.3                                                                           CH.sub.3                                                                           68                         ______________________________________                                    

The first entry provides the P₅₀ value obtained for a control of humanRBCs alone. The next two entries provide the P₅₀ values when the RBCsare mixed together with a 10 millimolar (mM) solution of the sodium saltof either 2-4-((((3,5-diclorophenyl)amino)carbonyl)methyl)phenoxy!-2-methylpropionic acid (C₁₈ H₁₇ Cl₂ NO₄) (discussed in Example 10) or 2-4((((3,5-dimethylphenyl)amino)carbonyl)methyl)phenoxy!-2-methylpropionic acid (C₂₀ H₂₃ NO₄) (discussed in Example 15), respectively.Note that the P₅₀ values for the hemoglobin in intact RBCs treated withthe compounds is much greater than the P₅₀ value for untreatedhemoglobin under physiological conditions (e.g., the control=27). Inaddition, it was determined that the P₅₀ value was raised from 27 to 31in the presence of 1 mM 2-4((((3,5-dimethylphenyl)amino)carbonyl)methyl)phenoxy!-2-methylpropionic acid and to 42 in the presence of 2 mM 2-4((((3,5-dimethylphenyl)amino)carbonyl)methyl)phenoxy!-2-methylpropionic acid. This data establishes the permeability of the compoundsto the cell membrane and that serum albumin does not interfere with thedrug's influence on the oxygen dissociation curve of hemoglobin. Thelast two entries in Table 2 provide the P₅₀ values for intact RBCstreated with 10 mM of the same two compounds, except that the RBCs werewashed with a 240 fold excess of 0.9% saline. The relatively slight dropin the P₅₀ value after the saline wash, which represents a highretention of allosteric effect, shows that the compounds used in thepresent invention have high binding affinity for hemoglobin.

FIG. 8 is a graph illustrating the oxygen dissociation curves producedwhen a 5.4 millimolar solution of normal hemoglobin is tested at pH 7.4using HEPES as the buffer in a Hem-0-Scan oxygen dissociation analyzer.As described above, the P₅₀ values reported in Table 1 were determinedfrom curves like those shown in FIG. 8. With particular reference toFIG. 8, the percent oxygen saturation (S O₂ on the vertical axis) isplotted against the partial pressure of oxygen (P O₂ on the horizontalaxis). Curve number 1 shows the oxygen dissociation curve (ODC) in theabsence of an allosteric modifying agent. Curve number 2 shows the ODChas been shifted to the right when 10 mM bezafibrate (a known rightshifting agent) solubilized with an equimolar amount of NaHCO₃ is addedto the hemoglobin. It should be noted that as the curve is right shiftedto a lower oxygen affinity state, the P₅₀ value increases. Curve number3 shows the right shift caused by adding a 10 mM concentration of 2-4-((((4-chlorophenyl)amino)carbonyl)methyl)phenoxy!-2-methyl propionicacid (C₁₈ H₁₈ ClNO₄) (described in Example 8 above) to the hemoglobin.Curve number 4 shows the right shift caused by adding a 10 mMconcentration of 2-4-((((3,5-dimethylphenyl)amino)carbonyl)methyl)phenoxy!-2-methylpropionic acid (C₂₀ H₂₃ NO₄) (described in Example 15) to thehemoglobin. Finally, curve number 5 shows the right shift caused byadding a 10 mM concentration of 2-4-((((3,5-dichlorophenyl)amino)carbonyl)methyl)phenoxy!-2-methylpropionic acid (C₁₈ H₁₇ Cl₂ NO₄) (described in Example 10) to thehemoglobin. The right shifting effect shown in FIG. 8 indicates thecompounds may be used to lower the oxygen affinity of hemoglobin.

FIG. 9 illustrates the effect of particular compounds on the ODC ofwhole human blood. Like FIG. 8, the percent oxygen saturation is plottedagainst the partial pressure of oxygen. As described above, the P₅₀values reported in Table 1 were determined from curves like those shownin FIG. 9. For these curves, 50 μl of whole human blood was mixed with a50 μl solution of the test compound in HEPES buffer at pH 7.4. Curvenumber 1 shows the ODC of hemoglobin in unreacted whole blood. Curves 2and 3 respectively illustrate the right shifting effect of the salts ofa 10 mM concentration of 2-4-((((3,5-dichlorophenyl)amino)carbonyl)methyl)phenoxy!-2-methylpropionic acid (C₁₈ H₁₇ Cl₂ NO₄) (described in Example 10 and referredto as RSR-4) or a 10 mM concentration of 2-4-((((3,5-dimethylphenyl)amino)carbonyl)methyl)phenoxy!-2-methylpropionic acid (C₂₀ H₂₃ NO₄) (described in Example 15 and referred to asRSR-13) on hemoglobin in whole blood.

FIG. 10 shows ODC curves of human hemoglobin (5.4 mM) in HEPES buffer atpH 7.4 which were made in a manner similar to that described inconjunction with FIG. 8. Like FIGS. 8 and 9, the percent oxygensaturation is plotted against the partial pressure of oxygen. Curvenumber 1 shows ODC of human hemoglobin in the absence of any allostericmodifying agent. Curves 3 and 5 show the right shifting effect of 1 mMand 10 mM concentrations of 2-4-((((3,5-dimethylphenyl)amino)carbonyl)methyl)phenoxy!-2-methylpropionic acid (C₂₀ H₂₃ NO₄) (described in Example 15 and referred to asRSR-13) on human hemoglobin. Hence, this compound forces hemoglobin to alower oxygen affinity state. Curve number 2 shows the right shiftingeffect of 2.5 mM 2,3-diphosphoglycerate (2,3-DPG), which is a naturalallosteric hemoglobin effector. Curve number 4 shows the combined effectof two effectors, e.g., 1 mM 2-4-((((3,5-dimethylphenyl)amino)carbonyl)methyl)phenoxy!-2-methylpropionic acid and 2.5 mM 2,3-DPG, is greater than either effectoralone. The synergistic effect may be utilized such that smallerquantities of drugs according to the present invention are added toblood.

Table 3 shows the utility of 2-4-((((3,5-dimethylphenyl)amino)carbonyl)methyl)phenoxy!-2-methylpropionic acid (called RSR-13) in preserving the oxygen affinity ofhemoglobin in stored blood.

                  TABLE 3                                                         ______________________________________                                        Packed RBC P.sub.50  in presence of RSR-13                                    Days old   0 mM         1 mM    2 mM                                          ______________________________________                                        Fresh      38           --      --                                            40         32           39      45                                            50         33           39      45                                            60         34           40      47                                            70         35           39      50                                            ______________________________________                                    

RSR-13, 1 mM and 2 mM, was added to samples of human RBCs (packed cells)which were stored at 4° C. in standard adsol formulation for 40-70 days.As can be seen from Table 3, the ODC of untreated blood left-shifts overtime (indicated by a drop in the P₅₀ value) to a high oxygen affinitystate. The increase in oxygen affinity of stored blood is attributed toa decreased concentration of 2,3-DPG. The P₅₀ value of 40 day olduntreated samples left shifted to 32; however, samples treated with 1 mMRSR-13 remained relatively unchanged (P₅₀ =39) and those treated with 2mMRSR-13 were right shifted (P₅₀ =45). Table 3 shows similarconcentration dependent effects of RSR-13 on the ODCs of 50, 60, 70 dayold packed cells. Because of the glycolytic metabolism, the pH ofuntreated red cells dropped over a period of time from 6.85 at 40 daysto 6.6 for 70 day old samples and this would possibly explain the slightright shifting of untreated 70 day old samples compared to 40 day oldsamples under the Bohr effect. The pH of red blood cells treated withRSR-13 was consistently lower than untreated samples, which suggeststhat RSR-13 favorably decreases the rate of glycolytic metabolism.RSR-13 had no adverse effect on the stability of RBCs as evidenced byconsistent RBC counts in treated and untreated samples. Similarly, theamount of hemolysis was consistent in both treated and untreated samplesof packed cells.

FIG. 11 shows the percentage oxygen delivered, ΔY, by packed cells.Changes in the oxygen saturation ΔY was calculated by Hill's equation(discussed in Stryer, Biochemistry, W. H. Freeman and Co., SanFrancisco, 1975, Chapter 4, pp, 71-94, which are herein incorporated byreference) at 100 to 30 torr. Column 1 shows the ΔY (59) correspondingto the untreated packed red blood cells. Column 2 shows the ΔY (50) ofpacked red blood cells stored for 40 days at 4° C. in the best availableadsol formulation. Column 3 shows that the ΔY=58 for 40 day old packedcells treated with RSR-13 (1 mM), which is comparable to fresh packedcells. Note, that the decrease (approximately 10%) in the oxygendelivery by packed cells is corrected by the addition of 1 mmol RSR-13.

Table 4 shows the change in the P₅₀ values of outdated packed red bloodcells on treatment with 2-4-((((3,5-dimethylphenyl)amino)carbonyl)methyl)phenoxy!-2-methylpropionic acid (RSR-13).

                  TABLE 4                                                         ______________________________________                                        Packed RBC P.sub.50  in presence of RSR-13                                    Days old   0 mM         1 mM    2 mM                                          ______________________________________                                        Fresh      38           --      --                                            40         32           38      42                                            50         31           38      45                                            60         34           39      46                                            ______________________________________                                    

50 μl of 40, 50, and 60 day old red cells were mixed with 50 μl ofRSR-13 to give final concentrations of RSR-13 at 1 mmol and 2 mmol.Control samples were prepared by mixing 1:1 packed cells and buffer. Ascan be seen from Table 4, the P₅₀ value of untreated samples wereconsistently lower than samples treated with RSR-13. In addition, acomparison of the results for the fresh red cells with red cells whichwere aged 40, 50, and 60 days shows a sharp decline in P₅₀ value withage. The P₅₀ values of 40, 50, 60 day old red cell samples treated with1 mmol RSR-13 were comparable to the P₅₀ =38 value found for fresh redcells. These results show that the addition of RSR-13 to the stored redcells restores the cells oxygen affinity.

FIG. 13a shows the chemical structure of bezafibrate (BZF). BZF is aknown antilipidemic drug which has been shown to decrease the oxygenaffinity of blood (see, Perutz et al., Lancet, 1983 (881), and Abrahamet al., Proc. Natl. Acad. Sci., USA, 1983, 80, 324). BZF possesses afour atom chain separting two aromatic units. Lalezari et al., J. Med.Chem., 1989, 32, 2352, have shown that shortening the four atom bridgebetween the two aromatic rings of BZF to a three atom urea linkage, asis shown in FIG. 13b, results in an increase in allosteric activity.However, the compounds shown in FIGS. 13a and 13b have been shown to beinactive at physiological concentrations of serum albumin.

Modified hemoglobin that gives up oxygen more readily has potentialsignificance in a wide variety of applications including emergencytransfusions, in radiosensitization of tumors, in increasing the shelflife of stored blood, and in the treatment of disease states caused byan insufficient supply of oxygen such as ischemia and hypoxia. Asdiscussed above in conjunction with Tables 1-4 and FIGS. 8-11, thecompounds of the present invention have been shown to possess allostericactivity in the presence of serum albumin and are, thus, usefulclinically. Specifically, the allosteric effectors of this inventionhave been shown to readily cross the red cell membrane in the presenceof serum albumin solutions, restore to normal the oxygen equilibriumcurves of outdated blood, shift the oxygen dissociation curves to theright in whole blood and in vivo in rats, and are not inhibited fromentering erythrocytes in the presence of the anion channel blockingagent DIDS.

FIGS. 14a-r show generallized chemical structures of several groups ofcompounds within the practice of this invention which have beensynthesized and tested. Contrasting the structures, it can be seen thatthe central bridge between the aromatic groups have been systematicallyrearranged, that modifications of the aromatic groups have been made,and that there are differences in substitution on the aromatic groups.For example, FIGS. 14a-e show chemical entities wherein the centralbridge includes NH, CO, and CH₂ moieties, FIG. 14f shows a chemicalentity with O, CO, and CH₂ moieties, FIG. 14g shows a chemical entitywherein part of the bridge between the aromatic end groups forms part ofa phthalimide compound, FIGS. 14h-j and 14q show chemical entities witha four atom bridge between the aromatics which include NH, CO, and twoCH₂ moieties, FIG. 14K shows a chemical entity with a central bridgewhich includes an amide moiety, FIG. 14L shows a chemical entity whereone of the aromatic groups is pyridine, FIGS. 14m-o show chemicalentities where the central bridge has varying numbers of atoms and wherethere are varying degrees of unsaturated conjugation (alkenes andalkynes) in the central bridge, FIG. 14p shows a two atom centralbridge, and FIG. 14r shows a bridge between the aromatics that includesa sulfur moiety.

All compounds depicted in FIGS. 14a-r, as well as the allostric modifiercompounds described above in conjunction with Tables 1-4 and FIGS. 1-7and 12, fall within a broad family of compounds having the generalstructure:

    R.sub.1 --(A)--R.sub.2

where R₁ and R₂ each are a substituted or unsubstituted aromatic orheteroaromatic compound, or substituted or unsubsituted alkyl orheteroalkyl ring compound, or a substituted or unsubstituted phthalimidecompound, and where R₁ and R₂ may be the same or different, where A is achemical bridge which includes two to four chemical moieties bondedtogether between R₁ and R₂, wherein said chemical moieties in A areselected from the group consisting of CO, O, S, SO₂, NH, NR₃ where R₃ isa C₁₋₆ alkyl group, NR₄ where R4 includes two carbonyls as part of aphthalimide compound formed with R₁ or R₂, CH₂, CH, and C, with thecaveat that, except in the case where A contains two identical CH and Cmoieties positioned adjacent one another to form an alkene or alkyne,the chemical moieties in A are each different from one another, andwherein at least one of R₁ or R₂ is substituted with a compound havingthe chemical formula: ##STR4## where n is zero to five, where R₅ and R₆are selected from the group consisting of hydrogen, halogen, substitutedor unsubstituted C₁₋₁₂ alkyl groups, carboxylic acids and esters,substituted or unsubstituted aromatic or heteroaromatic groups, andthese moieties may be the same or different, or alkyl moieties of partof an aliphatic ring connecting R₅ and R₆, and where R₇ is a hydrogen,halogen, salt cation, metal, or substituted or unsubstituted C₁₋₆ alkylgroup.

The protein-bound conformations and the hemoglobin binding sites of theallosteric modifier compounds of this invention were also determinedcrystallographically. The important interactions of the allostericmodifiers were determined to involve a salt bridge interaction of theacid group with Arg 141 α, a polar interaction of the amide carbonylwith Lys 99 α, and a hydrophobic interaction of the halogenated aromaticring with Phe 36.

The synthesis pathways for the chemical entities shown in FIGS. 14a and14b are described above. Examples 34-45 disclose the synthesis of thechemical entities shown in FIGS. 14c-r.

EXAMPLE 34

FIG. 15 shows a reaction scheme for preparing the 24-(arylacetamido)phenoxy!-2-methylpropionic acid compounds shown in FIG.14c. The syntheses were accomplished by reaction of the correspondingarylacetylchloride with 2(4-aminophenoxy)-2-methyl propionic acid in thepresence of a base. The intermediate 2(4-aminophenoxy)-2-methylpropionic acid was prepared from acetamidophenol.

EXAMPLE 35

FIG. 16 shows a reaction scheme for preparing the 24-((benzylamino)carbonyl)phenoxy!-2-methylpropionic acids shown in FIG.14d. The reaction requires suitably substituted benzylamine and4-hydroxybenzoic acid in the presence of PCl₅. The reaction provided theamidophenol in almost quantitative yield. The amidophenol was thenconverted to an isobutyric acid derivative by treatment with chloroformand acetone in the presence of sodium hydroxide. The compounds of FIG.141 which include a pyridine heteroaromatic moiety are prepared by asimilar process except that a 6-hydroxy 3-nicotinic acid is used insteadof 4-hydroxybenzoic acid.

EXAMPLE 36

FIG. 17 shows a reaction scheme for preparing the 23-(((arylamino)carbonylmethyl)phenoxy-2-methylpropionic acid compoundsshown in FIG. 14e. A suitably substituted aniline, 3-hydroxyphenylaceticacid, and PCl₅ in mesitylene were refluxed to afford the intermediateamidophenol. The reaction of intermediate amidophenol with CHCl₃,acetone, in base afforded the desired isobutyric acid. The analogs ofFIGS. 14a and 14k were prepared following a similar procedure asdescribed above, using 4-hydroxyphenylacetic acid and N-methylaniline,respectively.

EXAMPLE 37

FIG. 18 shows a reaction scheme for preparing the 24-((arylacetamido)methyl)phenoxy!-2-methylpropionic acids shown in FIG.14f. The reaction involved reacting a suitably substitutedarylchloroformate with 2 4-aminophenoxy!-2-methylpropionic acid in thepresence of base to provide the desired isobutyric acids.

EXAMPLE 38

FIG. 19 shows a reaction scheme for preparing the 2-4-(((phthalamido)N-methyl)phenoxy!-2-methyl propionic acids shown inFIG. 14g. The compound was prepared by refluxing phthalic anhydride and2 4-((amino)methyl)phenoxy!-2-methylpropionic acid in toluene in thepresence of triethylamine. The 2-4-(((phthalamido)N-methyl)phenoxy!-2-methyl propionic acid was obtainedas a crystalline white solid in 90% yield.

EXAMPLE 39

FIG. 20 is a reaction scheme for preparing the 2-4-(((arylamino)carbonyl)ethyl)phenoxy!-2-methylpropionic acids shown inFIG. 14h. A suitably substituted aniline, 4-hydroxyphenylpropionic acidand PCl₅ are refluxed in mesitylene for two hours to furnish theamidophenol. The amidophenol was then converted to the correspondingacid by reaction with acetone, chloroform, and base. The 2-4-(((benzylamino)carbonyl)methyl)phenoxy!-2-methylpropionic acids ofFIG. 14i were prepared by following a similar procedure, as is shown inFIG. 21, but using benzylamine instead of aniline and4-hydroxyphenylacetic acid instead of 4-hydroxypropionic acid.

EXAMPLE 40

FIG. 22a shows that the reaction of substituted arylacetylchloride with2- 4-(aminomethyl)phenoxy!-2-methylpropionic acid in the presence ofbase produces the 2- 4-((arylacetamido)methyl)phenoxy!-2-methylpropionicacid compounds of FIG. 14j. FIG. 22b shows a similar reaction schemewherein the four atom bridge compound shown as FIG. 14q is prepared. Thereaction procedure involves a conversion of a suitably substitutedphenylpropionic acid to the corresponding acid chloride by using thionylchloride in toluene. This acid chloride is then treated at 0° C. with analkaline (NaOH) solution of 2- 4-aminophenoxy!-2-methylpropionic acid inTHF and the reaction mixture is stirred for 2 hrs. Evaporation of THFleads to the recovery of the compound depicted in FIG. 14q.

EXAMPLE 41

FIG. 23 shows a reaction scheme for preparing the unsaturated conjugatedcompounds shown in FIG. 14m. A mixture of acetophenone and4-hydroxybenzaldehyde in ethanol and in the presence of 10% excesssodium hydroxide was heated to 70° C. to produce the intermediate,unsaturated keto alcohol. On treatment with acetone and chloroform inthe presence of base, the compound of FIG. 14m resulted.

EXAMPLE 42

FIG. 24 shows a reaction scheme for preparing the acetylene compoundsshown in FIG. 14n. A propiolic acid was converted to its acid chlorideby heating it with thionyl chloride. The acid chloride was then treatedwith 2- 4-(aminophenoxy)-2-methylpropionic acid at 0° C. in the presenceof base to produce the compound of FIG. 14n.

EXAMPLE 43

FIG. 25 shows a reaction scheme for preparing the unstaturatedconjugated four atom bridge compound shown in FIG. 14o.4-hydroxycinnamic acid was converted to the acid chloride by usingthionyl chloride and then the acid chloride was treated with aniline inp-xylene to produce the intermediate alcohol. The alcohol was convertedto the corresponding isobutyric acid derivative shown in FIG. 14o byreaction with acetone, chloroform, and a base.

EXAMPLE 44

FIG. 26 shows a reaction scheme for preparing the two atom bridgecompound shown in FIG. 14p. A mixture of suitably substituted anilineand 4-hydroxybenzoic acid using a catalytic quantity of PCl₅ inmesitylene were heated to 140° C. to produce the amidophenol, which wasthen converted to the correspoding isobutyric acid derivative of FIG.14p. Similar compounds can be produced by using3,5-dimethyl-4-hydroxybenzoic acid instead of 4-hydroxybenzoic acid.

EXAMPLE 45

FIG. 27 shows a reaction scheme for preparing the compound shown in FIG.14r which contains a three atom bridge containing the --SO₂ NHCH₂ --moiety. This is a one pot reaction involving a suitably substitutedaromatic sulphonyl chloride and 2-4-aminoethylphenoxy!-2-methylpropionic acid. The reaction is generallycarried out in THF at 0° C. in the presence of base.

Several compounds which fall within the groups defined by FIGS. 14a-rwere tested using a hemoglobin oxygen dissociation analyzer according tothe techniques and procedures described above. The compounds exhibitedallosteric effector activity by having a demonstrable "right shifting"in the oxygen dissociation curve. Table 5 summarizes the P₅₀ data forthe new derivatives wherein the ΔP₅₀ represents the difference in theP₅₀ measured for sample with analog and the P₅₀ for control. Since theshift in P₅₀ is dependent on the drug/hemoglobin ratio, all comparisonswere performed at a ratio of 4/1 analogue/Hb (i.e., 10 mM analogue/2.7mM Hb). The high Hb molarity (2.7 mM) used in the oxygen equilibriumstudies was intended to approximate the red cell Hb concentration (5mM). The P₅₀ measurements for BZF and the Lalezari compound of FIG. 13bare provided for comparison purposes.

                                      TABLE 5                                     __________________________________________________________________________    EFFECT OF ARYLOXY-2-METHYLPROPIONIC ACIDS ON THE                              OXYGEN AFFINITY OF HEMOGLOBIN                                                 Compound.sup.a                                                                      R.sub.2.sup.b                                                                     R.sub.3                                                                          R.sub.4                                                                           R.sub.5                                                                          R.sub.6                                                                          W  X  Y   Z  ΔP.sub.50                           __________________________________________________________________________    BZF--13a                                                                            H   H  Cl  H  H  CO NH CH.sub.2                                                                          CH.sub.2                                                                         15                                        13b   H   Cl H   Cl H     NH CO  NH 47                                        14a (1).sup.c                                                                       H   Cl H   Cl H     NH CO  CH.sub.2                                                                         64                                        14a (2)                                                                             H   Me.sup.d                                                                         H   Me H     NH CO  CH.sub.2                                                                         56                                        14a (3)                                                                             H   Me H   H  H     NH CO  CH.sub.2                                                                         8                                         14a (4)                                                                             H   H  OMe H  H     NH CO  CH.sub.2                                                                         34                                        14a (5)                                                                             OMe H  H   H  H     NH CO  CH.sub.2                                                                         32                                        14a (6)                                                                             H   OMe                                                                              H   H  H     NH CO  CH.sub.2                                                                         34                                        14a (7)                                                                             H   Br H   H  H     NH CO  CH.sub.2                                                                         24                                        14a (8)                                                                             H   OMe                                                                              OMe OMe                                                                              H     NH CO  CH.sub.2                                                                         39                                        14a (9)                                                                             H   Me Me  H  H     NH CO  CH.sub.2                                                                         40                                        14a (10)                                                                            ADAMANTYL.sup.c     NH CO  CH.sub.2                                                                         14                                        14a (11)                                                                            INDANYL.sup.f       NH CO  CH.sub.2                                                                         46                                        14a (12)                                                                            NAPHTHYL.sup.g      NH CO  CH.sub.2                                                                         40                                        14k   H   Cl H   H  H     NMe                                                                              CO  CH.sub.2                                                                         6                                         14b   H   Cl H   Cl H     CO NH  CH.sub.2                                                                         29                                        14c (1)                                                                             H   H  H   H  H     CH.sub.2                                                                         CO  NH 16                                        14c (2)                                                                             H   Cl H   H  H     CH.sub.2                                                                         CO  NH 26                                        14c (3)                                                                             H   Me H   H  H     CH.sub.2                                                                         CO  NH 27                                        14c (4)                                                                             H   H  Cl  H  H     CH.sub.2                                                                         CO  NH 25                                        14c (5)                                                                             H   H  F   H  H     CH.sub.2                                                                         CO  NH 17                                        14c (6)                                                                             H   H  Me  H  H     CH.sub.2                                                                         CO  NH 27                                        14c (7)                                                                             H   H  CF.sub.3                                                                          H  H     CH.sub.2                                                                         CO  NH 24                                        14c (8)                                                                             H   H  OMe H  H     CH.sub.2                                                                         CO  NH 20                                        14c (9)                                                                             H   H  Cl  Cl H     CH.sub.2                                                                         CO  NH 32                                        14c (10)                                                                            H   Me H   Me H     CH.sub.2                                                                         CO  NH 33                                        14d (1)                                                                             H   H  H   H  H     CH.sub.2                                                                         NH  CO 34                                        14d (2)                                                                             H   H  Cl  H  H     CH.sub.2                                                                         NH  CO 9                                         14d (3)                                                                             H   H  Me  H  H     CH.sub.2                                                                         NH  CO 9                                         14d (4)                                                                             H   Cl Cl  H  H     CH.sub.2                                                                         NH  CO 10                                        141.sup.h                                                                           H   Cl H   H  H     CH.sub.2                                                                         NH  CO 14                                        14f (1)                                                                             H   H  H   H  H     O  CO  NH 16                                        14f (2)                                                                             H   H  Cl  H  H     O  CO  NH 15                                        14f (3)                                                                             H   H  F   H  H     O  CO  NH 12                                        14f (4)                                                                             H   H  OMe H  H     O  CO  NH 11                                        14f (5)                                                                             H   H  Me  H  H     O  CO  NH 7                                         14f (6)                                                                             H   H  NO.sub.2                                                                          H  H     O  CO  NH 24                                        14f (7)                                                                             H   Me H   Me H     O  CO  NH 8                                         14h (1)                                                                             H   H  H   H  H  NH CO CH.sub.2                                                                          CH.sub.2                                                                         10                                        14h (2)                                                                             H   H  Cl  H  H  NH CO CH.sub.2                                                                          CH.sub.2                                                                         12                                        14i (1)                                                                             H   H  H   H  H  CH.sub.2                                                                         NH CO  CH.sub.2                                                                         12                                        14i (2)                                                                             H   H  Cl  H  H  CH.sub.2                                                                         NH CO  CH.sub.2                                                                         18                                        14j (1)                                                                             H   H  H   H  H  CH.sub.2                                                                         CO NH  CH.sub.2                                                                         6                                         14j (2)                                                                             H   H  Me  H  H  CH.sub.2                                                                         CO NH  CH.sub.2                                                                         19                                        14n   H   H  H   H  H  C  C  CO  NH 38                                        14o   H   H  Cl  H  H  NH CO CH  CH 12                                        14m   H   H  H   H  H     CO CH  CH 5                                         14p (1)                                                                             H   H  H   H  H        NH  CO 12                                        14p (2)                                                                             H   Cl H   Cl H        NH  CO 19                                        14p (3)                                                                             H   Cl Cl  H  H        NH  CO 14                                        14p (4)                                                                             H   Cl Cl  Cl H        NH  CO 18                                        14p (5)                                                                             H   Me H   Me H        NH  CO 17                                        14p (6)                                                                             H   Me Me  H  H        NH  CO 13                                        14q (1)                                                                             H   H  H   H  H  CH.sub.2                                                                         CH.sub.2                                                                         CO  NH 13                                        14q (2)                                                                             H   H  Cl  H  H  CH.sub.2                                                                         CH.sub.2                                                                         CO  NH 12                                        14r (1)                                                                             H   H  H   H  H     SO.sub.2                                                                         NH  CH.sub.2                                                                         4                                         14r (2)                                                                             H   H  Me  H  H     SO.sub.2                                                                         NH  CH.sub.2                                                                         8                                         14r (3)                                                                             H   H  Cl  H  H     SO.sub.2                                                                         NH  CH.sub.2                                                                         11                                        14r (4)                                                                             H   H  Br  H  H     SO.sub.2                                                                         NH  CH.sub.2                                                                         9                                         14r (5)                                                                             H   H  F   H  H     SO.sub.2                                                                         NH  CH.sub.2                                                                         8                                         14r (6)                                                                             Cl  H  H   Cl H     SO.sub.2                                                                         NH  CH.sub.2                                                                         10                                        __________________________________________________________________________     .sup.a Compound identifies structure shown in a particular drawing figure     identified by drawing figure number and letter.                               .sup.b R identifies substituents around on the left most aromatic moiety      indicated in the drawing.                                                     .sup.c number in parantheses reflects a variation in the aromatic region      where bridge region is identical.                                             .sup.d methyl                                                                 .sup.e admantyl moiety present in place of the left most aromatic moiety      indicated in the drawing.                                                     .sup.f Indanyl moiety present in place of the left most aromatic moiety       indicated in the drawing.                                                     .sup.g Naphthyl moiety present present in place of the left most aromatic     moiety indicated in the drawing (Naphthyl and phenyls are both aromatic).     .sup.h right most aromatic is pyridine.                                  

For exemplary purposes FIG. 14P shows substitution on the aromatic ringcontaining the propionic acid side chain. It should be understood thatall of the compounds within the family of compounds defined by thisinvention can have substitution on the aromatic ring containing thepropionic acid side chain and that the propionic acid side chain can belocated at any position on the aromatic ring. These compounds areprepared by, for example, using a suitably substituted 4-hydroxybenzoicacid such as 4-hydroxy 3,5-dimethylbenzoic acid or vanillic acid. Table6 discloses P₅₀ data for several different compounds defined by the FIG.14P structure.

                  TABLE 6                                                         ______________________________________                                         ##STR5##                                                                     R.sub.2                                                                            R.sub.3                                                                              R.sub.4                                                                              R.sub.5                                                                            R.sub.6                                                                           R.sub.7                                                                            R.sub.8                                                                             R.sub.9                                                                             R.sub.10                                                                           ΔP.sub.50             ______________________________________                                        H    CH.sub.3                                                                             H      CH.sub.3                                                                           H   H    CH.sub.3                                                                            CH.sub.3                                                                            H    19                          H    Cl     H      Cl   H   H    CH.sub.3                                                                            CH.sub.3                                                                            H    23                          H    CH.sub.3                                                                             H      CH.sub.3                                                                           H   H    OCH.sub.3                                                                           H     H    21                          ______________________________________                                    

While many of the compounds described above include a propionic acidside chain on one of the aromatic compounds, the side chain can bevaried to include halogens, C₁₋₁₂ alkyl groups, substituted andunsubsituted aromatic and heteroaromatic groups, or aliphatic ringgroups. Examples 29-32, above, set forth specific synthesis routes forobtaining these types of compounds. Table 7 presents the P₅₀ data forseveral compounds which have been synthesized that have the generalstructural formula: ##STR6##

                                      TABLE 7                                     __________________________________________________________________________    R.sub.2                                                                          R.sub.3                                                                          R.sub.4                                                                          R.sub.5                                                                          R.sub.6                                                                          R.sub.7                                                                            R.sub.8 R.sub.9                                                                              ΔP.sub.50                            __________________________________________________________________________    H  CH.sub.3                                                                         H  CH.sub.3                                                                         H  CH.sub.2 CH.sub.3                                                                  CH.sub.2 CH.sub.3                                                                     COOH   18                                         H  Cl H  Cl H  CH.sub.2 CH.sub.3                                                                  CH.sub.2 CH.sub.3                                                                     COOH   12                                         H  CH.sub.3                                                                         H  CH.sub.3                                                                         H  H    H       COOH   3                                          H  Cl H  Cl H  H    H       COOH   -1                                         H  CH.sub.3                                                                         H  CH.sub.3                                                                         H  H    CH(CH.sub.3).sub.2                                                                    COOH   24                                         H  CH.sub.3                                                                         H  CH.sub.3                                                                         H  H    CH.sub.2 C(CH.sub.3).sub.3                                                            COOH   19                                         H  CH.sub.3                                                                         H  CH.sub.3                                                                         H  CYCLOBUTYL.sup.a                                                                           COOH   27                                         H  CH.sub.3                                                                         H  CH.sub.3                                                                         H  CYCLOPENTYL  COOH   42                                         H  Cl H  Cl H  CYCLOPENTYL  COOH   11                                         H  CH.sub.3                                                                         H  CH.sub.3                                                                         H  CYCLOHEXYL   COOH   24                                         H  Cl H  Cl H  CYCLOHEXYL   COOH   17                                         H  CH.sub.3                                                                         H  CH.sub.3                                                                         H  CH.sub.3                                                                           CH.sub.2 CH.sub.3                                                                     COOH   24                                         H  Cl H  Cl H  CH.sub.3                                                                           CH.sub.2 CH.sub.3                                                                     COOH   17                                         H  CH.sub.3                                                                         H  CH.sub.3                                                                         H  CH.sub.3                                                                           COOCH.sub.2 CH.sub.3                                                                  COOCH.sub.2 CH.sub.3                                                                 1                                          H  CH.sub.3                                                                         H  CH.sub.3                                                                         H  CH.sub.3                                                                           COOCH.sub.2 CH.sub.3                                                                  COOH   3                                          H  CH.sub.3                                                                         H  CH.sub.3                                                                         H  CH.sub.3                                                                           COOH    COOH   4                                          __________________________________________________________________________     .sup.a Ring structure connecting R.sub.7 and R.sub.8.                    

Since the compounds contemplated by this invention are capable ofallosterically modifying hemoglobin so that a low oxygen affinity "T"state is favored (right shifting the equilibrium curve), these compoundswill be useful in treating a variety of disease states in mammalsincluding humans where tissues suffer from low oxygen tension, such ascancer and ischemia. As pointed out by Hirst et al. in Radiat. Res.,Vol. 112, (1987), pp. 164, decreasing the oxygen affinity of hemoglobinin circulating blood has been shown to be beneficial in the radiotherapyof tumors. The compounds may be administered to patients in whom theaffinity of hemoglobin for oxygen is abnormally high. Particularconditions include certain hemoglobinopathies and certain respiratorydistress syndromes including respiratory distress syndromes in new borninfants aggravated by high fetal hemoglobin levels and when theavailability of hemoglobin/oxygen to the tissues is decreased (e.g., inischemic conditions such as peripheral vascular disease, coronaryocclusion, cerebral vascular accidents, or tissue transplant). Thecompounds may also be used to inhibit platelet aggregation and may beused for antithrombotic purposes and wound healing. Topical applicationcould be used for wound healing. In addition, the compounds may be usedto treat low oxygen related disorders in the brain such as Alzheimer'sdisease, depression, and schizophrenia. It may be desirable toadminister the compounds to a patient prior to and/or simultaneouslywith the transfusion of the treated whole blood or red blood cells inorder to avoid substantial variations in the hemoglobin oxygen affinitydue to dilution that occurs when the blood is administered.

The compounds can be added to whole blood or packed cells preferably atthe time of storage or at the time of transfusion in order to facilitatethe dissociation of oxygen from hemoglobin and improve the oxygendelivering capability of the blood. Preferably, the compounds would beadded in an amount of about 50 mg to 1 g per unit of blood (473 ml) orunit of packed cells (235 ml). When blood is stored, the hemoglobin inthe blood tends to increase its affinity for oxygen by losing2,3-diphosphoglycerides. As described above, the compounds of thisinvention are capable of reversing and/or preventing the functionalabnormality of hemoglobin which is observed when whole blood or packedcells are stored. The compounds may be added to whole blood or red bloodcell fractions in a closed system using an appropriate reservoir inwhich the compound is placed prior to storage or which is present in theanticoagulating solution in the blood collecting bag.

Administration to a patient can be achieved orally, by intravenous orintraperitoneal injection, or rectally by suppository where the dose andthe dosing regiment is varied according to individual sensitivity andthe type of disease state being treated.

Studies with mice have shown that a mg/kg/day dose of 2-4((((3,5-dimethylphenyl)amino)carbonyl)methyl)phenoxy!-2-methylpropionic acid (C₂₀ H₂₃ NO₄) (discussed in Example 15 (RSR-13)) givenintraperitoneally is well tolerated. In vivo experiments with rodentshave also shown that (1) i.v.,infusion of RSR 13 results in a decreasein cardiac output and regional flows which indicates that an isolatedincrease in tissue oxygen delivery may increase total and regionalvascular resistance, (2) in the rodent model of focal cerebral ischemia,administration of RSR-13 results in reductions in the volume ofinfarction, and (3) in rodent experiments done with middle cerebralartery occlusion, the administration of RSR-13 decreased infart size.

If the compounds are used for wound healing, the compounds couldadvantageously be applied topically directly to the wound area. Inaddition, the compounds can be mixed with blood external to a patient'sbody prior to and/or simultaneously with a transfusion. The compoundscan be administered in the pure form or in a pharmaceutically acceptableformulation including suitable elixirs, binders, and the like or aspharmaceutically acceptable salts (lithium, soidum, potassium, ammonium,alkaline metals, etc.) or other derivatives (esters, ethers, etc.). Itshould be understood that the pharmaceutically acceptable formulationsand salts include liquid and solid materials conventionally utilized toprepare injectable dosage forms and solid dosage forms such as tabletsand capsules. Water may be used for the preparation of injectablecompositions which may also include conventional buffers and agents torender the injectable composition isotonic. Solid diluents andexcipients include lactose starch, conventional disintegrating agents,coatings and the like.

The following Examples discuss particular uses and administration routesfor the allosteric hemoglobine modifiers of this invention.

EXAMPLE 46

Radiation Oncology. Solid tumors are oxygen deficient masses. Theallosteric effectors of this invention deliver more oxygen to tumors,which increases radical formation that increases tumor killing duringradiation. In animal experiments, transplanted tumors (FSa-IIfibrosarcoma tumor of C3H mice) were injected with the allostericeffector and 30-60 minutes later they were exposed to 5-6 Gy ofradiation. The process was repeated for five consecutive days. Tumorswere measured for regrowth delay and then excised to obtain viablesingle cells that were plated for clonogenic survival in vitro. Thetests in mice show that the allosteric effector RSR-13 increases growthdelay of irradiated tumors by 50%.

EXAMPLE 47

Hypothermia limiting or preventing hypoxia induced irreversivemyocardial damage. The allosteric effectors increase the efficiency ofoxygen delivery at low blood flow and low temperatures, thus having theability to prevent myocardial damage.

EXAMPLE 48

Resuscitation from hemorrhagic shock. The allosteric effectors maydecrease the number of red blood cells required for treating hemorrhagicshock by increasing their efficiency of oxygen delivery.

EXAMPLE 49

Wound Healing, diabetic ulcers, chronic leg ulcers, pressure sores,tissue transplants. Experiments have shown that the allosteric effectorsdelivery of oxygen to wound healing is important. Damaged tissues healfaster when there is better blood flow and increased oxygen tension. Inaddition, by increasing oxygen delivery to wounded tissue, theallosteric effectors may play a role in the destruction of infectioncausing bacteria.

EXAMPLE 50

Stroke. The allosteric effectors will be effective in delivering oxygento the brain, especially before complete occlusion and reperfusioninjuries occur due to free radical formation. In animal experiments,approximately 30% reductions in the volume of infarction result afterthe administration of RSR-13. The reduction in infarct area obtained inthe animal studies reduces concerns about free radical damage fromincreased oxygen delivery in vivo to ischemic brain tissue.

In addition, animal studies have been conducted where the efficacy ofthe allosteric effectors in cat brains was assessed by measuring thediameter of small and large arterioles through cranial windows underhypoxic and hypotensive conditions. The studies show that the infusionof the allosteric effectors of this invention reduces the expansion ofarterioles to nearer normal controls under both hypoxic and hypotensiveconditions.

EXAMPLE 51

Cardiovascular/Angina applications. The allosteric effectors of thisinvention should be capable of increased oxygen delivery to blockedarteries and surrounding muscles and tissues, thus relieving thedistress of angina attacks. The compounds may serve as antithrombolyticagents and decrease fibrinogen.

EXAMPLE 52

Alzheimer's Disease. One of the many symptoms of Alzheimer's disease isdecreased flow of oxygen to the brain. The allosteric effectorsconcentrate in red blood cells which allows enhanced delivery of oxygento all areas of the body, including the brain. Thus, the allostericeffectors of the present invention can be used to combat the symptom ofdecreased oxygen flow to the brain and the resulting deterioration ofthe brain.

EXAMPLE 53

Acute Respiratory Disease Syndrome (ARDS). ARDS is characterized byinterstitial and/or alveolar edema and hemorrhage as well asperivascular lung edema associated with hyaline membrane, proliferationof collagen fibers, and swollen epithelium with increased pinocytosis.The enhanced oxygen delivering capacity attributable to the allostericeffectors of this invention can be used in the treatment and preventionof ARDS by combatting lower than normal oxygen delivery to the lungs.

EXAMPLE 54

Use of allosteric effectors with micelles or for use with underwaterexploration. Micelles are synthetic lipophylic membrane like spheresthat are being intensively investigated for in vivo administration ofbiological materials. Soya lecithin is a common agent used in creatingmicelles within a fluid. The micelles protect encapsulated drugs orbiological materials from undesired metabolism, antibody detection, etc.Addition of the allosteric hemoglobin modifiers of this invention tomicelles which encapsulate hemoglobin will increase the delivery ofoxygen to tissues. Since the allosteric effectors of this inventionconcentrate in erythrocytes when administered in vivo in rats,incorporation of the allosteric effectors in a micelle whichencapsulates hemoglobin allows the effector to remain securely withinthe micelle until it has been degraded. In addition, because of theincreased delivery of oxygen attributed to the allosteric effectors ofthis invention, the allosteric effectors can be used to increase thedive time for underwater divers.

EXAMPLE 55

The in vivo effects on hemoglobin-oxygen (Hb-O₂) affinity and tissue PO₂were investigated after intraperitoneal administration of 2-4-(((dichloroanilino)carbonyl)methyl)phenoxyl!-2-methyl propionic acid(RSR-4; 150 mg/kg) or its 3, 5-dimethyl derivative (RSR 13; 300 mg/kg)in C3Hf/Sed mice. The Hb-O₂ dissociation curve was plotted from tailvein blood samples using an O₂ dissociation analyzer prior to and up to160 minutes after compound administration. Twenty to forty minutes afterinjection, the hemoglobin P₅₀ increased by a mean 25% (range 18-31%)after RSR 4 and 53% (range 36-76%) after RSR 13. Tissue PO₂ increased bya mean of 78% (range 30-127%) after RSR 4 and 66% (range 39=97%) afterRSR 13 administration in anesthetized mice. No change was observed intissue P O₂ in anesthetized controls.

While the invention has been described in terms of its preferredembodiments, those skilled in the art will recognize that the inventioncan be practiced with modification within the spirit and scope of theappended claims.

We claim:
 1. A compound of the general structural formula: ##STR7## where R₁ is selected from the group consisting of optionally substituted phenyl, adamantyl, naphthyl, and indanyl;where R₂ and R₃ are alkyl moieties of a C₃₋₆ alkyl ring connecting R₂ and R₃ ; and where R₅ is selected from the group consisting of hydrogen, C₁₋₃ lower alkyl, and a monovalent salt cation.
 2. The compound of claim 1 wherein R₁ is a substituted or unsubstituted phenyl.
 3. The compound of claim 1 wherein R₁ is a substituted or unsubstituted indanyl.
 4. The compound of claim 1 wherein R₁ is a substituted or unsubstituted adamantyl.
 5. The compound of claim 1 wherein R₁ is a substituted or unsubsituted naphthyl.
 6. The compound of claim 1 wherein R₂ and R₃ are alkyl moieties of a C₅ alkyl ring connecting R₂ and R₃.
 7. The compound of claim 1 wherein R₂ and R₃ are alkyl moieties of a C₆ alkyl ring connecting R₂ and R₃.
 8. The compound of claim 1 wherein R₂ and R₃ are alkyl moieties of a C₄ alkyl ring connecting R₂ and R₃.
 9. The compound of claim 1 where the moiety containing R₂, R₃ and R₅ grooves are connected at the para position of the phenyl ring.
 10. The compound of claim 9 where R₁ is substituted with a substituent selected from the group consisting of halogens, NO₂, C₁₋₃ alkyl, halogenated C₁₋₃ alkyl, C₁₋₃ ether, and C₁₋₃ carboxylic acid ester. 